Milk Proteins

MILK PROTEINS By H. A. McKENZlE Deportment of Physical Biochemistry, Institute of Advanced Studies, Australian National University, Canberra, A.C.T., Australia

I. Introduction.. . . . .

........................

................

IV. A Note on Heterogeneity, Association-

tion, and Conformation of

B. Reactions in Transport Experiments.. .

V. Caseins. . . . . . . . A. Isolation of B. Methodsof

........................

64

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67

....................

......................

E. us-Caseins... . . . . . . . . .

Major Whey Proteins.. ........................

.............

G . Serum Albumin.. ... B. Acid Phosphatase.. ......................

55

56

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H. A. MCKENZIE

B. Immunoglobulin Types in Milk.. ...................... C. Importance of Immunoglobulinsin Milk.. . . . . . . . . . . . . IX. Milk Proteins and Allergenicity Reactions. . . . . . . . . . . . . . . . . . A. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Milk Protein Antibodies in Serum.,. . ............ C. Allergens in Milk.. . . . . . . . . . . . . . . . . . ............ X. Future Research.. . . . . . . . . . . . . . . . . . ....... References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

216

221

I. INTRODUCTION Preparation of the mammary glands for lactation begins early in pregnancy, and secretion of milk begins soon after the end of gestation. The newborn mammal obtains its nourishment and some of its immunity from the milk produced by the maternal mammary glands. Production of milk is a specific mammalian adaptation, and milk is unique in being an almost complete natural food. Under natural conditions milk passes directly from mother to offspring. The capacity for lactation may be more variable in man than in animals because successful lactation is not necessary for the survival of man. Milk substitutes are readily available when a mother cannot or will not breast feed. The most common substitute is cow milk. Because of its excellent nutritive value, cow milk (and foods derived from it) has become used widely by humans of all ages. Nevertheless numerous problems arise in its use, preservation, and processing. It is not surprising that most attention has been given hy scientists to the properties and composition of cow milk rather than milk of other species. The composition varies from species to species, which is probably due to the marked variation in needs of the young. Moreover, the composition varies for a given species with the stage of lactation. Early studies of milk proteins were directed toward their nutritive and immunological properties. Revolutionary advances in the methods of protein chemistry have enabled advances in our understanding of protein structure and composition, dreamed barely possible twenty years ago. Thus scientists have been less daunted by the complexity of the milk proteins and have now made progress in understanding them. Present work is based firmly on the pioneering studies of Sprrensen and Sgirensen, Linderstrom-Lang, and McMeekin. Two discoveries in the last decade have done most to stimulate new work: the discovery by Aschaffenburg and Drewry (1955) that @-lactoglobulinis a mixture of genetically different proteins, and the discovery by von Hippel and Waugh of a casein component, K-casein, which is responsible for the micelle-stabilizing properties and is the component on which the enzyme rennin acts prior to the clotting process. When the milk proteins were last reviewed in Advances in Protein Chemistry and in The Proteins (McMeekin and Polis, 1949; McMeekin,

MILK PROTEINS

57

1954), neither of these discoveries had been made. In the last decade no major review has appeared on milk proteins as a whole. However, several reviews have appeared on the p-lactoglobulins and caseins. (These reviews will be considered in the sections on the individual proteins concerned.) The reader is referred for background information on the mammary gland and its secretion to the treatise of Kon and Cowie (1961). The unique significance of milk proteins in mammalian nutrition and their immunological role alone justifies the study of the chemistry of these proteins. However, they possess physicochemical properties that make them of great importance in protein chemistry. The individual proteins undergo an array of interactions with themselves and with one another. They exhibit a variety of conformations. The study of their associationdissociation reactions and conformations poses many problems. In the present review, special attention is given to these physicochemical properties and attention is drawn to the pitfalls involved in their study. The significanceof the problems in the study of proteins in general is emphasized. The reviewer endeavors to give an overall picture of the present state of knowledge of the chemistry of milk proteins. The field is so wide and the volume of published work in the last decade so enormous that it has become virtually impossible for one person to deal authoritatively with every facet of milk proteins. Thus, while the reviewer has endeavored to examine every paper he has found listed in major abstracting journals (up to December 1965), he has found it neither expedient nor desirable to discuss and cite every such reference. Naturally he has tended to give more space to those aspects with which he is most familiar and to emphasize areas he considers of most importance. Some milk protein chemists may not agree with this assessment; however, it is hoped that they will at least be stimulated to further work. Protein chemists working in other areas will be able to obtain from many of the sections a general picture, and to see the enormous implications of G l k proteins in our overall knowledge of protein structure.

11. COMPOSITION OF MILK The opalescent appearance of mature milk is so striking that the word “milky” is frequently applied to other media when it is intended to convey a description of opalescence or cloudiness. This characteristic appearance of milk arises from the presence of micelles of the caseins, the principal protein group of milk, The casein micelles incorporate other milk constituents: they provide an excellent example of how, in considering particular proteins, the protein chemist must take careful account of their environment and their interactions with it. Caseins are present in milk not only in the large aggregates characteristic of micelles, but also with

58

H. A. MCKENZIE

TABLE 11-1 Approximate CompoSiliOn of Mature Bovine Milk ~

~

Constituents ~~

Approximate concentration (gm /I iter)

~

Water Proteins Caseins @-Lactoglobulin a-Lactalbumin Serum albumin Immunoglobulins Others (lactoferrin, lactollin, etc.) Fat globule protein

870 25 3 0.7 0.3

0.6 1.3

0.2

Enzymes Lactoperoxidase Acid phosphatase Alkaline phosphatase Protease Xanthine oxidase Amylase Lipases (esterases) Ribonuclease Lysozyme Carbonic anhydrase Lipids Fat Phospholipids Sterols Carotenoids Vitamin A Vit,amin D Vitamin E

0.3 0.1 0 . 3 x 10-8 0 . 3 x 10" 0.4 X lod 1 x 10"

Carbohydrates Lactose Other sugars

(small smt.)

Ions Calcium(I1) Magnesium(I1) Sodium (I) Potassium (I) Phosphates Citrates Chloride Bicarbonate Sulfate

40

50

1.3 0.1

0.5

1.5 2.1 2.0 1.0 0.2 0.1

59

MILK PROTEINS

TABLE 11-1 (Continued)

Constituents Vitamins Inositol Choline Ascorbic acid Thiamine Riboflavin Niacin Pyroxidine Pantothenic acid Biotin Folk acid Vitamin B I ~ Nitrogenous (NPN) Ammonia (N) Amino acids (N) Urea (N) Creatine (N) Uric acid Uracil-4-carboxylicacid Hippuric acid Indican Phosphate esters Trace elements Rb, Li, Ba, Sr, Mn, Al,Zn,B,Cu, Fe, Co, I Gases (milk exposed to air) Carbon dioxide Oxygen Nitrogen

Approximate concentration (gm/liter). 180 x 10-8 150 X 10-8 20 x 10-8

0.4

x 10-3

1.5 x 0.7 x 0.7 x 3.0 X 50 x 1x 7x

10-8

10”

10-8 10-6 10-6

10-

7 x 10-8 3 x 10-8 100 x 10-8 15 X lo-’ 7 x 10-8 70 x 10-a 50 x 10” 1 x 10-8 0 . 1 x 10-3

100 x 10-8 8 X lo-* 15 X lowa

particle weights in a range more characteristic of the common globular proteins in aqueous solution. Immediately after parturition the total protein content of bovine colostral milk (colostrum) is high (even as high as 200 gm/liter) and the major proteins present are not caseins, but immunoglobulins, the ratio of immunoglobulin to casein being as high as 2 : 1. The immunoglobulin content falls very rapidly and is soon less than the casein content, which also falls but much more slowly. Although bovine milk is more or Iess “normal milk” in ca. 4 days after parturition, the trends evident in the colostrum period continue for some weeks through a period of transitional milk until the phase of mature milk is reached.

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H. A. MCKENZIE

The total protein content of mature milk varies considerably among individual cows and varies among breeds, and there are seasonal variations (see, e.g., Rolleri et al., 1956; Larson and Kendall, 1957). There are also variations in the amounts of individual proteins. An interesting example is the correlation found by Aschaffenburg and Drewry (195713) between the amount of casein produced by an individual cow and the amount and kind of &lactoglobulin. The total protein content of mature milk is of the order of 31 gm/liter of which ca. 25 gm/liter is casein. While attempts have been made in the past to estimate the concentration of individual proteins in milk, they have been based mostly on methods that cannot give precise values, due mainly to inadequate resolution. With the improvements achieved in sensitivity of physical and immunological methods fordetermination of heterogeneity and the development of techniques of isolation of proteins, it should now be possible to make far more precise estimates. Most of the noncasein proteins of inilk are whey proteins and enzymes associated primarily with the aqueous phase. However, small amounts of proteins and enzymes are associated with the lipids (which are of the order of 40 gm/liter). While we shall be concerned primarily in this review with milk proteins and enzymes, and interested in other constituents only insofar as they are known to play a role in the behavior of the proteins and enzymes, it is useful to have before us a reminder of the overall composition of milk. Such data for bovine milk are summarized in Table 11-1, adapted from that of Jenness and Patton (1959). 111. MILKPROTEIN NOMENCLATURE The nomenclature for milk proteins has long been a matter of some controversy. During the last decade many new milk proteins have been discovered, and workers have not been slow to give a new (real or imagined) . protein they isolated a name. Thus it seemed that something approaching chaos was fast developing on the horizon, especially as a number of names proposed appeared to be possibly different names for the same protein in various states of purity or even degradation. A responsible body in the United States, the American Dairy Science Association, set up committees to report on the nomenclature of milk proteins. The first report was issued in 1956 (Jenness et al., 1956); this was followed by a revision in 1960 (Brunner et al., 1960) and more extensive revision in 1965 (Thompson et al., 1965). The latter report is a comprehensive and careful revision by a group of distinguished workers. Their report deserves careful and sympathetic consideration. The task of developing a rational classification for milk proteins, covering both present and possible future proteins, is far from an easy one.

MILK PROTEINS

61

The reviewer considers that any recommendations for the nomenclature of milk proteins should take careful cognizance of current terms and concepts in protein chemistry as a whole. There has been a tendency among workers on milk proteins to continue to use some terms now outmoded and in some cases downright misleading. Also, some proteins in milk are synthesized in the udder; others are transferred from the blood and appear to be identical with the corresponding protein in blood. There Seem to be little justification for giving the latter proteins names different from those applicable to the corresponding blood protein. The reviewer accepts the majority of recommendations of the A.D.S.A. Committee. However, he asks milk protein chemists to give their earnest consideration to a number of suggestions he makes for improved nomenclature. The following is a summary of the nomenclature used in this review. Where the reviewer's usage departs from the A.D.S.A. recommendation, this is indicated clearly and reasons are given for departure from the A.D.S.A. nomenclature. Casein. In 1838 Mulder showed that a protein could be precipitated from bovine milk by the addition of acid. A method for the purification of this protein, casein, after precipitation with acetic acid, was published by Hammersten in 1900. Both the definition of casein and the method of preparation were like Topsy, they just grew. It is amazing that a century passed after Mulder's work before real use was made of two important properties of casein in order to prepare it without adjustment of the pH value from neutrality, namely, its interaction with calcium(I1) and its precipitation in the presence of high concentrations of neutral salts, e.g., (NHJ2S04. Despite alternative methods of preparation being available, an operational definition of casein in terms of acid precipitation is usually employed: whole casein is the protein precipitated from skim milk at pH 4.6. The A.D.S.A. definition is more precise: '(a heterogeneous group of phosphoproteins precipitated from skim milk at pH 4.6 and 2 0 O . " It is fortunate that the A.D.S.A. has specified the temperature, for different precipitates are obtained a t various temperatures in the range 0-37", commonly used for acid precipitation of casein. Some workers have precipitated their casein a t pH 4.6 and 2". We shall see (Section V) that precipitation is far from complete at pH 4.6 and 2". Even a t 20' the precipitation is not complete in the opinion of the reviewer, and the casein carries with it other proteins (e.g., transferrin, protease). Furthermore, the casein appears to be altered in some way by the acid precipitation a t the higher temperatures. During the alkali dissolution of acid casein, all or part of the y-casein may be lost (El-Negoumy, 1963; Lahav, 1965). There are difficulties in proposing alternative definitions. The calcium precipitation method of von Hippel and Waugh (1955) results in a prepara-

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H. A. MCKENZIE

tion that is obtained under very mild conditions, but all the casein does not seem to be isolated by this method. For some years the reviewer and his colleagues have been investigating the isolation of whole casein from milk by ammonium sulfate fractionation. There are many advantages (as discussed in Section V) of using this gentle reagent in protein fractionation, and these apply equally well to casein. The question immediately arises as to what one is separating out by this salt fractionation. It seems that virtually all the casein is separated out, but in addition some of the so-called ‘‘proteose peptone,” immunoglobulins, and some glycoproteins containing phosphorus are included. It is proposed below that the “proteose peptone” is a ca.sein and that this undesirable term should be dropped. The amount of the immunoglobulins in mature milk is small but significant. Along with these come one or more glycoproteins containing phosphorus. The reviewer cannot see why the latter should be rejected out of hand as being caseins, Nobody knows a t present just what role these proteins play in milk and whether they contribute to the stability of the micelle. There certainly seems to be no less justification at present, for including such protein as a casein than for including y-casein. The reviewer considers it preferable to start with a preparation that contains all the “caseins” prepared in a gentle fashion together with a small amount of “noncasein proteins,” which are removable in subsequent fractionation, than to start with a preparation that does not contain all the “casein,” that may be partly altered, and that contains some noncasein proteins. All workers in reporting their research should state very clearly how they prepared their whole casein and what part, if any, wm thrown down the “drain.” In 1939 Mellander first observed the resolution of acid casein by movingboundary electrophoresis at pH 8.6 into three peaks, which he designated a , (3, and y in order of decreasing mobility. In view of what we currently know about the interpretation of transport patterns, it does not follow that each of these peaks is necessarily related to a protein component. However, it is possible to develop methods of fractionation that give a-,@-,and y-fractions, which, when separately subjected to electrophoresis, give peaks corresponding in mobility to the a-,p-, and y-peaks, respectively, of whole casein. We know now that each of these fractions is a mixture of proteins, and that different methods of preparation do not give identical fractions (see Section V). Nevertheless it is convenient to refer to many of the casein components in terms of these fractions. It was considered originally that a-casein was a single protein and that it was the fraction upon which the enzyme rennin acted. Waugh and von Hippel (1956) showed that casein micelles contain a complex of a-casein and another casein they called K-casein. They considered that, K-casein is

MILK PROTEINS

63

the fraction on which rennin acts primarily, and it is soluble in the presence of calcium(I1) whereas the a-casein is sensitive to calcium(I1). Subsequently the calcium-sensitive a-fraction has been referred to as a,-casein (Waugh, 1958), apcasein, aR-casein (Long et al., 1958), wcasein (McMeekin et al., 1959), and a-caseins 1.07 and 1.10 (Schmidt and Payens, 1963). Thompson et al. (1962) reported the discovery of three genetic forms of as-casein, which differed only slightly in mobility in urea-starch gel electrophoresis. The A.D.S.A. Committee has rightly pointed out that a nomenclature scheme that will embrace these and possible future variants is difficult to formulate. They designate the aa-casein fraction as that fraction of a-casein precipitated by calcium(I1) a t 0-4’ and stabilized by K-casein in the presence of calcium(I1). They recommend that the genetic variants (A, B, and C) be designated as asl-A,ael-B,aSl-C. I t is recommended also that the genetic designations should be accompanied by figures for the relative mobilities of each variant in the urea-starch gel-buffer system of Wake and Baldwin (1961). This operational definition is not without problems: it is based on an empirical electrophoretic procedure, and it is difficult to fit possible future variants that do not differ in electrophoretic mobility into it. However, it is the best one suggested to date. In this review we shall consider K-casein to be the principal casein upon which rennin acts, to be soluble in the presence of 0.40 M calcium(I1) a t pH 7.0 and 04”,and to stabilize aB-caseinagainst precipitation by calcium. The A.D.S.A. Committee defines fi-casein in terms of its solubility in 3.3 M urea at pH 4.6 and its temperaturedependent polymerization. Unfortunately neither of these properties is characteristic of @-caseinalone. It should be possible to define it rather more satisfactorily if a specification regarding its mobility in electrophoresis were added to the other specifications. The y-casein fraction has also been defined in terms of urea solubility (insolublein 3.3 M urea, soluble in 1.7 M urea, a t pH 4.7, with (NH&S04). The heterogeneity of each of these casein fractions will be discussed further in Section V. The whey proteins fraction of milk has often been referred to as the “lactalbumin fraction.” The latter term is unsatisfactory and should be replaced by the term whey protein (also used by the A.D.S.A. Committee). It is recommended that there be a prefix to indicate the method of preparation, e.g., acid whey proteins, ammonium sulfate whey proteins, rennin whey proteins. The nomenclature for the a-lactalbumins, @-lactoglobulins, and serum albumin is fairly straightforward. The recent discovery of a new @-lactoglobulin in the milk of Droughtmaster beef cattle by Bell et al. (1966a)

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H. A. MCKENZIE

means that a separate nomenclature from that for dairy breeds may have to be adopted for such 0-lactoglobulins in case additional variants are discovered in the dairy breeds. The p-lactoglobulin concerned has been Bell and Stormont (1965) have obnamed 8-lactoglobulinDr,,ht,..t,,. tained the first evidence of genetic variants of serum albumin in milk. Their work indicates that there are variants of equine milk serum albumin. Suitable nomenclature will be needed for them. The nomenclature of the iron-binding proteins of milk has been far from satisfactory. At least one type of these proteins is milk specific and at least one other type is common to the blood serum of the animal. The former will be termed Zactoferrins. The latter will be classified as transfewins, a term proposed by Holmberg and Laurel1 (1947) to denote a specific function. The reviewer’s nomenclature is given in Section VI,E. The antibody fractions of milk have been referred to frequently as These terms will “ lactoglobulins,” “euglobulins,” and “ pseudoglobulins.” not be used in this review. The proteins concerned will be referred to as immunoglobulins and the nomenclature proposed by a committee of the World Health Organization (Ceppellini et al., 1964) will be followed for individual immunoglobulins. While this nomenclature was designed principally for human immunoglobulins, it can be applied to those of other species provided the criteria recommended by Ceppellini et al. are applied. The nomenclature of Heremans et al. (1963) and one of the older designations (see discussion in Putnam, 1965) will be given in parentheses for the convenience of the reader. In Section V it will be shown that the major components of the so-called “proteose peptones” of milk are probably components of casein. Thus it is considered that this misleading term should not be used and that the components should be designated by more suitable names. The nomenclature for the enzymes of milk is comparatively straightforward and follows standard enzyme nomenclature.

IV. A NOTEON HETEROGENEITY, ASSOCIATION-DISSOCIATION, AND CONFORMATION OF MILK PROTEINS A . Introduction The problems of the chemistry of milk proteins are common to those of other proteins: the determination of their state of occurrence, their interactions with one another, their heterogeneity with respect to size and chemical composition, their primary structure, and their molecular architecture. While the earliest criteria used for determining heterogeneity of milk proteins were based on phase solubility tests, transport experiments have

65

MILK PROTEINS

been used most commonly. Some thirty years ago Pedersen (1936) examined the rate of sedimentation of the proteins in skim milk after dialysis against phosphate buffer. His sedimentation diagram showed a considerable number of peaks (Fig. IV-1). The complexity of interpretation is increased when it is realized that a given peak does not necessarily represent a monomeric or polymeric form of a single protein, let alone a chemically homogeneous protein. The same kinds of consideration apply to other transport experiments, e.g., moving-boundary and zone electrophoresis and column chromatography.

0.15

ato

0 55

6.0

6.5

7.0

FIG.IV-1. Sedimentation pattern of skim milk dialyzed against phosphate buffer, pH 6.8 (0.02M KHzPO,, 0.03 M Na2HP04,0.2 M NaCl). The abscissa represents distances from the center of rotation, and the ordinale represents scde line displacements that are proportional to the concentration gradient. Sedimentation is from left to right. Relative centrifugal force: 260,000 g. Exposure: 13 min after reaching speed. The a,@-pe& arises primarily from a-lactalbumin and p-lactoglobulin. The Speak and peaks (From Pedersen, 1936.) to the right of it arise mainly from -in.

Svedberg and Pedersen (1940) realized early that many proteins (e.g., hemoglobin and hemocyanin) were able to undergo association-dissociation reactions. It is surprising how slow biochemists have been to realize the importance of such phenomena in the behavior of proteins and enzymes. During the last fifteen years the reviewer has stressed their importance in his lectures to both undergraduates and experienced workers in the field. He offers no apology for stressing it again in the current context. While Svedberg’s old multiplicity hypothesis was incorrect, there is an element of truth in this concept in that the majority of proteins and enzymes appear to undergo an array of polymerizations of comparatively small units, each unit being specific for a given protein or enzyme (McKen~ie,1960). Small genetic differences in these units can have a profound effect on the protein’s

66

H. A. MCKENZIE

association-dissociationbehavior, as is illustrated in the section on the @-lactoglobulinsin the present review. Before proceeding to discuss these reactions it is important to indicate the use given in this review to certain terms, especially as some of them are used in a somewhat different way from that used by certain polymer and colloid chemists. Proteins may be regarded as being formed by the “polymerization” of amino acids. The amino acid residues are linked through the peptide bond to form a chemical entity that may consist of one chain or more than one chain covalently linked. This minimum molecular weight entity will be called the protein “monomer unit.” This term is preferred by the reviewer to the term “subunit” used by many workers (see Lacks et al., 1964). These minimum molecular weight units may undergo association to form polymers (or “n-mers”) through noncovalent linkages of the types discussed by Kauzmann (1959). The degree of polymerization is frequently small, but sometimes large aggregates may be formed leading to “micelles,” precipitates, or gels. The term, micelle, is used here to refer to a charged colloidal aggregate or particle. This is in the original sense of the word as used by von Ntigeli (1858) and even by the colloid chemist McBain. In discussion a t the Faraday Society Meeting in 1913 on colloids McBain remarked, “NOWtake some of these highly charged colloidal aggregates, micelles, or ‘colloidal ions’ we are discussing.” In a later paper (McBain and Salmon, 1920) he pointed out that he had introduced the concept of “micelle” at the 1913 Discussion I‘ to remove one of the chief difficulties in interpreting the properties of acid and alkali albumin.” At first sight transport experiments, in which each monomer or n-mer moves with its own characteristic velocity, might seem to be an easy way to detect association-dissociation. However, Tiselius (1930) realized that, if interconversion occurs sufficiently rapidly to maintain the equilibrium during the transport experiment, the boundary between solvent and solution will not reflect the properties of the separate constituents of the solution, but some mean of them, Alexander and Johnson (1949) considered association reactions in which (i) the times required for establishment of equilibrium is long compared with the transport experiment, (ii) the equilibrium time is comparable with that of the experiment, and (iii) the equilibrium time is small compared with that of the experiment. They concluded, inter alia, that in the latter case a single discrete peak will be observed. Thus it is not surprising that protein chemists received a shock when Gilbert (1955; see also Gilbert, 1959, 1963; Gilbert and Jenkins, 1959) showed that, for associationdissociation reactions of type (iii), one, two, or more peaks were possible in transport experiments. The identification of

MILK PROTEINS

67

the number of peaks observed with the number of components present becomes a hazardous occupation. The milk protein chemist needs to know how to design experimenh to detect such reactions and to be able to detect them in the work of others.

B. Reactions in Traneport Experiments 1. General We shall now consider briefly the way in which experimental patterns in transport processes are affected by interactions of the type

A and B may be similar protein molecules (polymerization reaction) or unlike molecules (reaction to form a “complex”). The reactions may be classified with respect to rate in the manner of Alexander and Johnson above. The reader is referred for detailed reviews to the recent articles of Nichol et a2. (1964), Nichol and Ogston (1965), and Nichol (1965). Transport experiments, involving mass flow under the influence of a potential gradient, may be divided broadly into two types. The first type is represented by: (a) “zone” electrophoresis in a supporting medium such as filter paper, cellulose acetate membrane, starch, acrylamide, or agarose gel; a band of protein solution is applied a t the origin and, after application of a voltage gradient for a period, a band or series of bands of proteins are obtained, and (b) chromatography in which a small zone of protein solution is applied to the top of a column of chromatographic material, e.g., DEAEcellulose or Sephadex, with subsequent elution to obtain an elution profile. The latter is a plot of concentration versus tube number or elution volume. In this type of experiment, if the initial zone consists of a noninteracting mixture of proteins, the individual species will separate, providing resolution is adequate, into a series of discrete peaks. However, if the initial zone of protein solution contains polymerizing species in rapid equilibrium, the zone will spread as it moves down the column as a result of chemical reaction and diffusion. Ultimately there may possibly be no position in the zone where the total concentration equals the initial concentration. Since the dilution due to spreading favors dissociation, the final pattern obtained may give a misleading picture of the relative amount of monomer and n-mer. For certain purposes, this difficulty with the first type of experiment can be overcome by use of a second type of experiment. In the latter the experiment is arranged to include a region where the total concentration

68

H. A. MCKENZIE

is the same as the initial concentration throughout the duration of the experiment. In the frontal analysis technique of chromatography (Claesson, 1946), sufficient solution is applied to the column so that, when the leading front emerges, it is followed by solution in which the protein concentration equals the initial concentration. The protein is then eluted in the usual way, giving a trailing edge. There is a close analogy between this type of experiment and moving-boundary electrophoresis and sedimentation velocity experiments. The situation in sedimentation velocity and the descending edge in moving-boundary electrophoresis are analogous I

I

A I B

Distance I

Ascending

Leadink

t

c moving-boundary electrophoresis + descending*

zone chromatography with plateau region t trailing

FIG.IV-2. Plots of concentration (c) arid concentration gradient (ac/az) versus distance (z)for a tranbport experiment involving a plateau region in which two nonreacting species, A and B, exist: * Equivalent to sedimentation velocity boundary.

to the trailing edge in the plateau chromatographic experiment-in all three cases protein moves away from the buffer region toward the plateau region. The situation in the ascending limb in moving-boundary electrophoresis is analogous to the leading edge in the chromatographic experimentprotein is moving away from the plateau region into buffer. The concentration gradient versus “distance” plot in each case is of similar type (see Fig. IV-2). It is to be contrasted with the type of concentration versus “distance” plot pertinent to the usual type of zone electrophoresis or chromatography experiment. In making the above analogy it is assumed implicitly that the relative order of magnitude of the velocities of all the

MILK PROTEINS

69

species is the same in all the transport processes considered (e.g., v, > v,-~ in a polymerization reaction, which may not hold for d l kinds of process). 2. Slow Reactions

If the time of establishment of equilibrium is long compared with the time required for separation of species in transport experiments of the above type, then the following features will be seen in the plot of ac/ax vs. 2:

(a) There will be a single boundary for each molecular species present in the protein solution. The value of &/ax will fall to zero between peaks, if the mobility difference between the species and the cell or column length are both sufficiently great to permit resolution. (b) The corrected area of each discrete peak (see Johnston and Ogston, 1946) provides a measure of the equilibrium concentration of each species in the initial solution. (c) The ascending and descending patterns, when observed, will approach being enantiographic, in marked contrast to a rapidly reacting system. (d) The patterns of a slowly reacting system should be distinguishable from those of simple mixtures of proteins. The effect of dilution with various times of standing before sedimentation, the effect of temperature, and group-specific reagents for intermolecular bonds should be examined. 3. Rapid Reactions

Gilbert (1955, 1959) showed that, for a rapidly equilibrating monomer n-mer system, where n = 2, one asymmetrical peak would be observed in the transport experiment for the plot of &/ax vs. 2,as shown in Fig. IV-3. An example of this type of behavior is given by the monomer-dimer system of P-lactoglobulin (the pattern, of course, being rounded due to diffusion), and is discussed in Section VI. Gilbert showed also that, if n > 2 and only monomer and a single n-mer are present, a bimodal pattern, of the type shown in Fig. IV-3, may be obtained. If a series of experiments is carried out in which the total protein concentration is varied, a n interesting result is obtained. At very low concentration a single peak only appears. As the concentration increases the absolute area of this peak increases until the second fast-moving peak (if v, > v,) appears, and thereafter the abso2ute area of the original peak remains constant. Eventually, a t high concentration, its relative area with respect to the other peak becomes quite small. I n such a system the value of ac/& never falls to the base-line value between the peaks. Furthermore, neither peak represents pure monomer nor pure n-mer, and neither peak moves with the velocity of pure monomer nor pure n-mer. I n moving-boundary 'electrophoresis the ascending and

70

H. A. MCKENZIE

descending patterns of such a system are highly nonenantiographic. All these features provide obvious tests to determine whether a given system is of this type. If n is > 2 it is extremely likely, from the general principles of reaction mechanism, that stepwise formation of intermediate polymers occurs. Very few such cases have been worked out in detail, but it can be concluded from them that such systems can give a single peak or exhibit multimodality. The “ tetramerization” of &lactoglobulin near pH 4.6 exhibits interesting features of the Gilbert theory, and can be treated reasonably satisfactorily as though “monomer” and (‘tetramer” (actually dimer and octamer, see Section VI) only are present.

Position

FIQ.IV-3. Theoretical plots of concentration gradient (ac/az) versus distance (2)for sedimentation velocity pattern of rapidly equilibrating systems: (a) monomer $ dimer, (b) monomers n-mer (n > 2) without intermediates. (Modified from Gilbert, 1955.)

The concentration dependence of the velocity of the peaks in associatingdissociating systems is characteristic. In Gilbert’s earlier theory, the effect of concentration on the sedimentation coefficient and the effect of diffusion were neglected. In his more recent work, allowance has been made for the former, and his calculated curves are shown in Fig. IV-4 along with the experimental values of Timasheff and Townend (1961 a). (This concentration dependence is discussed further in Section VI.) Rapid reactions of the A B C need separate treatment from polymerization reactions (nA A,). Gilbert and Jenkins (1959) and more recently Nichol and Ogston (1965) have given theoretical treatments for a number of reactions of this type. The latter have also shown that the theory is applicable to physical as well as chemical interactions. This work has considerable implications in the study of milk protein interactions, especially those of the caseins. It is outside the scope of the present review to deal with even the six cases dealt with by Gilbert and Jenkins (1959). The situation is complex and the reader is warned against generalizing from the examples given. Their paper is very valuable and the reader should use it as a guide in planning or interpreting experiments on milk proteins

+

71

MILK PROTEINS

I

2

4 ¢ralion

6 (gm/100 ml)

L

8

(0)

81

I

I

I

I

I

I

I

I

1

FIG.IV4. Concentration dependence of sedimentation coefficients ( ~ 2 0 . ~ of ) leading and trailing peaks of bovine p-lactoglobulin A at pH 4.65 (NaOAc-HOAc, Z O.l), 2": (4-, theory of Gilbert (1963); ---, earlier theory of Gilbert (1959); 0 , experimental points of Timasheff and Townend (1961a); (b) theoretical concentration , ~ bovine 8-lactoglobulin A under same conditions. Theoretical dependence of ~ 2 0 for curves assuming various amounts of nonassociating protein: (- --) 0 yo;(-) 10%; (---.) 33 %; (- -) individual species.

-

involving possible complex formation. Computed patterns for one of their examples are given in Fig. IV-5, where vc > V A > vB. I t will be noted that the trailing boundary represents pure reactant (A or R) depending on the values of K ( C A )and O K(cs)o, where K is the equilibrium constant for the reaction, and ( c A ) ~and ( c g ) o are the initial concentrations of A and B. For a given system ( K constant), variation of the initial mixing pro-

H.

A. MCKENZIE

E qTl-T (a)

k -I

-

b*

-1%t

0

I

2

3

“6

yn

yc

yn

yc

FIG.IV-5. Theoretical transport patterns for R complex C and its components, A and B; vc > V A > OB, X parameter of Gilbert and Jenkins = 5 . In (a), K ( C A ) O= 1.5, K ( c B ) ~ = 0.25; in (b), K ( C A ) = O 1.0, K ( C B ) = O 0.5. (From Nichol et al., 1964, after Gilbert and Jenkins, 1959.)

portions over a wide range will result in the replacement of a peak characteristic of one reactant by one related to the other reactant.

4. Reactions of Intermediate Rate Where the rate of attainment of equilibrium is comparable with that of the transport measurement, the pattern for such a system is complicated. Even for simple isomerization reactions of the type A B, one, two, or even three peaks may develop with increasing time (see Cann and Bailey, 1961; van Holde, 1962). These peaks cannot be identified obviously with individual protein species. 5 . Zone Transport Processes without Plateau Regions

The bands or peaks (corresponding to a c vs. x plot) obtained in the pattern correspond directly to individual components for a slow reaction, and there is no great difficulty introduced by such a reaction. If a, rapid reaction occurs, the final c vs. x plot will represent a reaction zone. Concentration patterns revealed by staining could well reveal two maxima in staining intensity superimposed on a blurred background. Similar pattern appearances could be obtained with reactions of intermediate rate. No compreheiisive theories are at present available for interpretation of zone

MILK PROTEINS

73

processes without plateaus. Thus zone processes with plateau are recommended in preference to those without plateau for investigation of milkprotein polymerizing systems. Zone transport processes nevertheless play an extremely important role in investigation of the heterogeneity of milk proteins, providing they are interpreted cautiously and each band is not ascribed to an actual protein component without further investigation. It is important to realize that an important practical test can be applied: protein material can be removed from a band in many instances and re-run through the transport process to determine if it again behaves as a single band. Cann and Goad (1965) have developed a theory of zone electrophoresis for systems of the type P

+ nHA

P(HA),

where P represents a macromolecular ion in solution and P(HA) I't s complex with n moles of an uncharged constituent, HA, of the solvent medium. It is assumed that up # uP(HA)% and that the equilibrium is established instantaneously. Numerical solutions of the conservation equations show that the zone electrophoretic pattern may resolve into two stable and intense zones. Other conditions may yield a single, broad, trailing zone. This work is very pertinent in the zone electrophoresis of milk proteins, where they are frequently examined in solvents containing boric acid. C . Weight Average Properties

Another experimental approach to the study of associatingdissociating systems is to determine a weight average property at a series of total concentrations of the protein. Provided sufficient time is allowed for the attainment of equilibrium and the solvent and temperature are kept constant, the weight average property should increase with increasing total protein concentration (alternatively, it should decrease on dilution) in contrast to that for a noninteracting system. The variation of weight average property with concentration enables the calculation of an equilibrium constant and n for the reaction and, if the reaction is also carried out over a range of temperatures, of the thermodynamic quantities A F O , AH", and AS". It will be shown in later sections that such determinations have been made successfully for milk Rroteins by light scattering, optical rotatory dispersion, sedimentation equilibrium, and the method of Archibald. If the effect of ionic strength, organic solvents, and temperature on the weight average property is determined, useful information may frequently be obtained on the type of noncovalent bonding involved in the aggregation (see Kauzmann, 1959). The application of these principles to casein and @-lactoglobulinis discussed in the relevant sections (V and VI).

74

H. A. MCKENZIE

D . Molecular Weights i n Dissociating Solvents The tendency of milk proteins to complex, associate, and aggregate causes the research worker to search continually for suitable solvents to dissociate them into their monomeric form, so that the protein monomer molecular weight may be determined. Usually a dissociating agent must be added to a solvent in fairly high concentration, if it is to be effective. This means that we have to deal with a multicomponent solvent, and problems of selective solvation, if not considered carefully, can result in considerable errors in the determination of the monomer molecular weight, even if the protein is completely dissociated, e.g., casein in a dissociating solvent. The increasing use of urea and guanidine hydrochloride of high concentration to dissociate milk proteins means that special attention must be paid to solvation or exclusion, as the density increment of these components is high (cf. Schachman, 1960). Sometimes a dissociating component of lower density increment can be chosen. The treatment of multicomponent systems has been discussed by Williams et al. (1958), Fujita (1962), and Casassa and Eisenberg (1964). The latter have proposed a definition of components, which is very valuable for many purposes (see also Scatchard and Bregman, 1959). If proper use is to be made of it, a lengthy dialysis of the protein against the solvent is required before carrying out the particle weight or virial coefficient measurement. Such a dialysis is neither practicable nor desirable for the study of some associating protein systems (e.g., where slow aggregations or conformational changes are occurring).

E. Conformation There are at present two main ways to gain information on the conformation of milk proteins: X-ray analysis of protein crystals, and optical rotatory dispersion or circular dichroism of protein solutions. The former method is capable of giving the detailed structure of a protein. This fantastic achievement!has been accomplished in the case of a few proteins, but as yet of no milk proteins, although work on &lactoglobulin is well advanced. In the few cases so far compared (hemoglobin, myoglobin, and lysozyme) the conformation of the protein in solution, as determined by optical rotatory dispersion, seems, under certain conditions, to be similar to that deduced from the X-ray measurements. Nevertheless proteins frequently display considerable flexibility in structure and this “motility” (a term used by the late K. Linderstrdm-Lang) is an important feature. Thus a note of caution must be sounded against making too many conclusions on the conformation of proteins in solution from work on crystals.

MILK PROTEINS

75

On the other hand, present measurements on optical rotatory dispersion of proteins are generally interpreted empirically, assuming there are only two structures present: helical and disordered (see the excellent review in Advances in Protein Chemistry by Urnes and Doty, 1961). Such a treatment has proved very useful for many proteins. It is satisfactory for some milk proteins, such as the caseins, which are probably primarily in random chain configurations. However, proteins such as the P-lactoglobulins may require the consideration of other structures if optical rotatory dispersion measurements are to be interpreted satisfactorily. Recent extension of optical rotatory dispersion measurements to the 190-mF region, hypochromism measurements of the 190-mp absorption band, and increasing development of equipment making possible circular dichroism measurements down to 190 mp are enabling more satisfactory estimates to be made of the efective helical content of proteins. Problems arising in the interpretation of such measurements have been discussed by a number of authors. The reviewer recommends especially the articles by Kauzmann (1957,1959), Urnes and Doty (1961), Schellman and Schellman (1964), Litman and Schellman (1965), and Holzwarth and Doty (1965).

V. CASEINS A . Isolation of Whole Casein

It was pointed out (Section I11 on "Milk Protein Nomenclature") that it is desirable to consider methods of preparation of whole casein other than the established acid precipitation method. During the last four years, McKenzie et al. (1962-1965) have compared a number of preparative procedures. They used milk from a single cow in most of their work. It is now known that the caseins from this cow contain the following genetic variants: a,,,-casein B/B, /?-casein A/R, and K-casein A/B. In all preparations the fat was removed from the milk by centrifugation at 1-3'. The methods of isolation that were compared are: (1) Acid precipitation at So". Hydrochloric acid (1 N ) is added with mechanical stirring to the skim milk at 30 f 2" over a period of 4 0 4 5 minutes until a pH value of 4.54.6 is attained. Stirring is continued for a further 30 minutes. The precipitate is sedimented at -1300 g, washed twice with water, and centrifuged. It is then dissolved by suspension in a volume of water ca. half that of the skim milk used and titrated with sodium hydroxide (1 N ) to pH 7.0-7.2. The precipitation, washing, and dissolution procedures are repeated. (2) Acid precipitation at 3'. The procedure is similar to that, in (l),

76

H. A. MCKENZIE

except that all operations are carried out at 2-3" and the acid is added until a pH value of 4.3 is attained. This pH value is used, as precipitation is by no means complete at pH 4.6 and 2". (3) Low temperature centrifugation in the presence of added caZcium(II)at So. The procedure is similar to the original procedure of Waugh and von Hippel (1956) for preparation of first cycle casein (see Section V,C), except that citrate is used instead of oxalate to remove calcium(I1). Three layers result in the centrifuge tube. A t the bottom there is a pale yellow opalescent gel, on top of this a small amount of an opalescent viscous liquid layer and the main supernatant. Each of these layers is removed and sodium citrate added to 0.1 M , and each is dialyzed initially against 0.1 M sodium citrate and then exhaustively against 0.1 M sodium chloride. (4) Low temperature centrifugation in the absence of added caZcium(l1)at 3". The procedure is the same as for (3), except that no calcium(I1) is added to the milk. ( 5 ) Ammonium sulfate precipitation at So. Ammonium sulfate (260 gm/liter) is added to the skim milk with mechanical stirring over a period of ca. 40 minutes. Stirring is continued for a further 75-90 minutes and the precipitate collected by centrifugation. The precipitate is washed with ammonium sulfate solution, recentrifuged, and dissolved in water (ca. half the volume of skim milk). The procedure is repeated and the final solution dialyzed exhaustively against glass-distilled water. (6) Ammonium sulfate precipitation at $0". The procedure is the same as for (5), except all operations (except the final dialysis) are conducted at 20" instead of 3". The general properties of these preparations are compared in Table V-1. The conclusions from this work may be summarized briefly as follows: (1) High speed centrifugat,ion of skim milk, in both the presence and absence of added calcium(II), results in a gel-like layer of protein, on which there is a small volume of a viscous liquid layer of protein, the amount of which is less when calcium(I1) has been added, The appearance of the gel is similar to that of the gel formed by centrifuging ammonium sulfateprecipitated casein at 2" or 20". Acid casein, when precipitated and centrifuged at low temperature (<4"),also forms an opalescent gel, but as the temperature of precipitation rises the character of the precipitate changes dramatically. Even at 5-6" there is an appreciable change, the precipitate being more granular. The precipitate at 30" is quite coarse, granular, and much less hydrated. It tends to settle out on standing, whereas the low temperature acid casein and the ammonium sulfate casein precipitates require moderate centrifugal forces to separate them out satisfactorily. The low temperature acid casein can be dissolved with some difficulty at pH 7 by addition of alkali, the 30" acid casein prepara-

MILK PROTEINS

77

tions being soluble with greater difficulty. Solution of acid casein by dialysis against a phosphate buffer of pH 7 ( I 0.1) can be achieved only with considerable difficulty. On the other hand, ammonium sulfate casein is readily soluble in dilute salt solution. Solution of the gel resulting from centrifugation of milk can be achieved reasonably easily by dialysis against 0.1 M sodium citrate (pH 6.8) and 0.1 M sodium chloride. (2) The yields of acid casein and ammonium sulfate casein are somewhat higher than those of the centrifuged casein, even when there is added calcium(I1) present. There is little difference in total nitrogen or phosphorus contents between the preparations. However, ammonium sulfate preparations with somewhat lower phosphorus contents have been obtained occasionally. (3) It is now well known that the sedimentation patterns obtained with solutions of whole casein are very dependent on the pH, temperature, ionic strength, and protein concentration. Some attempt was made to compare the sedimentation patterns of the various preparations a t a protein concentration of 10 gm/liter, temperature < 5 O , and I = 0.1. In most of the patterns there are two peaks, a fast, rapidly spreading peak of variable szo,w and a slow peak of fairly constant ~ ~ 0 ,It~ is. dangerous to ascribe any particular caseins to either of these peaks (see Section IV). Some workers consider the fast peak to be due mainly to a-K-caseins and the back peak mainly to p-casein. This is probably a considerable over-simplification. Certainly the back peak has an szo,w of about 1.3-1.5 S, which is consistent with the szo,w of monomeric p-casein. However, this peak is obviously heterogeneous, and it is possible that monomeric K-casein and other caseins are present in it. The szo,wvalue of the fast peak is variable, being lower in the phosphate buffer of pH 7.0 than in 0.1 M NaCl as solvent. There is also less polydispersity evident in the phosphate buffer, which could be related to sequestration of remaining traces of calcium(I1) in the casein preparations (cf. Philpot and Philpot, 1939). I t is evident that, in acid casein precipitated a t higher temperatures, considerable aggregation has occurred and disaggregation can be effected only with difficulty. After solution with the aid of alkali, dialysis against 0.1 M NaCl is not as effective in causing disaggregation as the pH 7.0 phosphate buffer. The relative areas of these peaks vary considerably in the various preparations, and are not completely reproducible for a given type of preparation from batch to batch of milk. No satisfactory explanation can be advanced for this variation at present, but it is obvious that many factors are affecting the polymerization and complexing of the casein components present. (4) Urea-starch gel electrophoretic examination of samples has been made by the method of Wake and Baldwin (1961) in both the presence and absence of mercaptoethanol. The function of the latter was to give sepa-

TABLEV-1 Properlies of Casein Preparaiionci Casein preparation Property ( aI

30",acid

2") acid

2", (N&)Zm4

Zoo, (N&)2%4

Calcium(X1)

Form of precipitate

Coarse, p n d a r ; little hydrated

Gel-like, slightly granular

Soft opalescent gel

Separation

1200 g, 30 min, some settling under g only Little affected by

37,000g, 50 rnin

14,600g, 35 rnin

Less gel-like after washing with Ha0

Dissolves in HZO, Dissolves in H20, Blend with NaCl or little affected by little affected by CaClz soh. (NH4)&04soh. (NI14)2SO~ soln. wash wash Remove Ca(II), Dissolves in H20, Dissolves in H20, complete in 50 complete in 50 dialyze exhausmin min tively

Washing

Dissolution

Reprecipitation, solution Yield (% total milk protein)

H20

Dissolves with aid Dissolves with aid of alkali; soh. of alkali; soln. (60 gm/l) with (60 gm/l); 90% difficulty; ca. 90% in 30 min, rest in 60 min, rest with with some diffigreat difficulty culty More granular, Slightly more more difficult to granular, more dissolve difficult to dissolve 81.3

Soft opalescent gel Soft opalescent gel viscous liquid layer 14,600 g. 35 min 78,480g, 90 min

+

Gel a little harder: Gel a little harder, Similar ppt., remove dissolves in H20 dissolves in HsO Ca(II), dialyze exhaustively

82.0

82.6

66.3

(a)

15.4 f 0 . 2

N content (gm/ 100 gm) P content (gm/ 100 gm) NANA (gm/ 100 P)

(4

(ii) A m (10 gm/l, 1 cm)

15.7 k O . l

(0.1 M NaCl, pH 6, 2-4', c = 10 gm/l)

15.4 f 0.1

15.2 f 0.2

0.79 f 0.05

0.77 k 0.05

0.75 f 0.05

0.74 f 0.05

0.80 -I 0.05 (gel)

0.41 f 0.02 0.60 -I 0.02 8 . 4 f 0.2

0.42 k 0 . 0 2

0.46 k 0.02 8.7 f 0 . 1 5

0.47 f 0.02 0.67 f 0.02 8.7 f 0 . 1 5

0.45 f 0.02 (gel) 8.1 f 0.2 (gel)

-

1.3 f 1 . 5 s 5.M.OS if pptd. at 30", fast 7 S 1.4-1.65 5.0-6.0 S

-

8.6 5 0 . 1

1.3-1.4s 1.2-1.4 S 1.4-1.5s 8.0-12.0s 7.0-9.0 S 5.8-6.5 S polydispersity very fast material S?O,W Slow 1.3-1.4s 1.3-1.5 S (NaCl, PO1, I = Fast 4.7-4.8 S 4.6-4.9s some very fast some very fast 0.1, pH 7, 2-4", c = 10 gm/l) material material 5 major bands 5 major bands 5 major bands Urea-starch gel 12-15 minor bands 12-15 minor bands 12-15 minor bands Electrophoretic pattern

s2o.v

2

15.4 f 0.2

Slow Fast

+

+

+

1.3-1.5 6 6.0 S 5 major bands 12-15 minor bands

5 major bands 12-15 minor bands

80

H. A. MCKENZIE

rate bands in the K-casein region. The citrate concentration used was half that of Wake and Baldwin and the gels were stained with the more sensitive nigrosiae. Overall the patterns were very similar. The patterns for acid casein preparations showed less intense staining of some of the slowest minor bands compared with those of the ammonium sulfate preparations. It is not yet certain whether this is due to lower concentration of K-casein components in the acid casein. (5) Several proteins are known to be associated in minor amounts with acid casein: lactoferrin (see Section VI,E), lactollin, acid and alkaline phosphatases, lipase, and a protease. No data have yet been obtained on the extent to which these occur in ammonium sulfate- and calcium(I1)precipitated caseins. McKenaie and Murphy found that the unequivocal detection of protease in casein preparations is not easy, as confusion with bacterial contamination and other reactions can easily occur. Zittle (1965) has made a number of important observations on the occurrence, detection, and purification of protease in casein (discussed in Section V,F). (6) Analysis for N-acetylneuraminic acid (NANA) of the gel, viscous liquid layer, and supernatant from the high speed centrifugation of milk, with and without added calcium(II), has given interesting results. Some 84 % of the casein is in the gel and viscous liquid layers, of which the NANA content is 0.43 gm/lOO gm protein. However, the NANA content of the supernatant liquid is 0.17 gm/100 gm. This means that ca. 12 % of the total NANA remains in the supernatant. However, this does not necessarily mean that 12 % of K-casein is in the supernatant. This amount of NANA cannot be ascribed to the immunoglobulins present in the supernatant from mature milk, as it is far too high for the amount of immunoglobulin present. It is concluded below that an important glycoprotein fraction of casein is not completely separated by this method. This conclusion is in accord with the interesting experiments of Sullivan et al. (1959) on the u-casein distribution in milk. The NANA content of preparations from milk of the same cows can vary appreciably, even during the mature milk phase; examples of this are given [under (ii) of the NANA content] in Table V-lb. The amount of NANA recovered in ammonium sulfate casein preparations is higher than for any of the other preparations. The major part of the difference cannot be ascribed to precipitation of immunoglobulins along with the casein. This matter is further discussed below. (7) Ammonium sulfate fractionation gives a good preparation of casein, although not without problems, such as the coprecipitation of minor impurities that require subsequent removal. It provides a gentle method of fractionation, offeriiig advantages that have been discussed generally by Dixon and Webb (1961).

81

MILK PROTEINS

M i n o r Phosphoglycoproteins The ammonium sulfate casein preparation contains most (if not all) of the “proteose peptone” fraction of milk. It is important to consider this fraction. Osborne and Wakeman (1918) appear to have been the first workers to describe such a fraction. They found that variable amounts of material remained in solution following acid precipitation of the casein and precipitation of the “albumins” and “globulins” by boiling the acid whey. They concluded that this fraction did not arise from hydrolytic degradation of the main milk proteins and that it was present to the extent of ca. 0.2 gm/liter. Later Rowland (1938) found that portions of the milk serum proteins were not rendered acid precipitable a t pH 4.6, following heat treatment of skim milk a t 95-100” for 30 minutes. This fraction could be divided into two fractions on the basis of solubility in 0.5 saturated ammonium sulfate solution. Aschaffenburg (1946) prepared a fraction from both acid whey and rennin whey by salting it out of the heat-cleared whey with ammonium sulfate. His fraction was completely insoluble in 0.5 saturated ammonium sulfate and hence does not conform to the original definition of a peptone. He called this fraction a-proteose on account of its marked surface activity. The behavior of u-proteose in moving-boundary electrophoresisexperiments indicated that it was heterogeneous. Ogston (in an addendum) examined the preparation in the ultracentrifuge. It was obviously polydisperse, only 60 % of the material appearing in the pattern, some 50 % with 8 2 0 , ~= 0.96 S and some 10 % with s20.w = 2.75 S. Subsequently Larson and Rolleri (1955) designated certain peaks in moving-boundary electrophoresis of acid whey as possibly being proteose peptones.” There was a small peak, “8,” with a mobility a little greater than that of serum albumin a t pH 8.6, a larger peak, “5,” with a mobility intermediate between that of a-lactalbumin and ,&lactoglobulin a t pH 8.6, and asmall peak, “3,” with a mobility a little less than that of a-lactalbumin but of the order of that of faster immunoglobulins. Aschaffenburg and Drewry (1959) observed that six bands were present in filter paper electropherograms of preparations made by the Rowland method. The major band corresponded to Larson and Rolleri’s peak “5,” and the five small bands to his peak “3.” The same six bands were found in concentrates prepared from ultrafiltrates of unheated milk. Thus the material causing these bands occurs in mature milk and does not require heating and acidification to produce it. Aschaffenburg found that he could prepare this heterogeneous fraction by salting out from acid whey with sodium sulfate to a concentration of 120 gm/liter. It appears from the ((

82

H. A. MCKENZIE

yellow stain given with bromphenol blue on the filter paper strips that all bands contained carbohydrate. Jenness (1959) found that he could precipitate a protein mixture containing material corresponding to peak “5” by saturating skim milk with sodium chloride. The precipitate was redispersed, casein removed at pH 4.6, the supernatant concentrated, lactoperoxidase adsorbed on an ion-exchange resin, and material corresponding to peak “5” obtained by selective precipitation at pH 4.5. Over 90 yoof this material had a mobility at pH 8.6 of -4.5 X low5cm2V-l sec-’. It contained 1.2-1.3 % P, was soluble in the presence or absence of 0.033 A4 calcium(II), and was not clotted by rennin. Evidence was obtained that it is the so-called loaf volume depressant. Brunner and Thompson (1961) compared the Rowland (1938), Aschaffenburg (u-proteose) (1946), Jenness (1959), and Weinstein et al. (1951) fractions and the soluble membrane protein fraction of Herald and nrunner (1957). All were heterogeneous in moving-boundary electrophoresis at pH 8.6 and a t pH 2.4, and showed considerable boundary spreading in sedimentation velocity pattern at pH 8.6 ( I 0.1). The soluble membrane protein had a major sedimentation peak of szO= 9 S and a minor peak of s20 = 18 S. All the other fractions had a peak of sz0 = 0.8-0.9 S and a second peak of s20 = 2.6-2.9 S, except the Jenness fraction where the second peak had sz0 = 5.9 S. All had N contents in the range 10-14 gm/100 gm protein, ash contents of 3-7 gm/100 gm, and phosphorus of 0.6-1.5 gm/100 gm and contained hexose sugars. Thompson and Brunner (1959) found their Rowland-type preparation to have a hexose content of 2.9 gm/100 gm,hexosamine 1.2 gm/lOO gm, fucose 0.7 gm/100 gm, and sialic acid 2.0 gm/100 gm. Kolar and Brunner (1965) were able to release material, with a mobility (-8 X cm2V-’sec-l, pH 8.6) similar to that of peak “8,” from casein micelles by heating, by addition of acid to pH 4.6, by action of rennin, by freezing, or by dehydration. They also isolated it from acid casein and from acid whey. Kolar and Brunner consider this material to be the tenacious contaminant” in K-casein preparations, and that it may be identical to the fraction called X-casein by Long et al. (1958). They found it to migrate as a single band in urea-starch and polyacrylamide gels. The value for s20,wat pH 7.0 (phosphate) is 1.0 S. The nitrogen content is 14 %, phosphorus 1.2 yo, and it contains hexose, hexosamine, and sialic acid. Marier et a2. (1963) found that the protein precipitated from mature skim milk by 0.73 M trichIoroacetic acid contains 17-28 % more sialic acid than does casein prepared by acid precipitation at pH 4.5. They isolated a fraction from the serum of heated milk that contained 1.8 gm sialic acid/

MILK PROTEINS

83

100 gm of the fraction. This component accounted for the above difference in sialic acid distribution. McKenzie and Murphy (1965) made preparations according to the methods of Aschaffenburg and Drewry (1959) and Rowland (1938). They found that the fraction of Aschaffenburg and Drewry had anitrogen content of 15.7 yo, a sialic acid content of 0.74 %, and sedimented in the ultracentrifuge to give a major peak of 1.4 S and a small amount (5 %) of a faster peak (3.8 S). The two fractions (soluble and insoluble a t 0.5 saturates (R7H4)2SO4) of the Rowland preparation had nitrogen contents of 15.6 % and 14.2 %, sialic.acid of 0.9 % and 2.4 %, respectively. The insoluble fraction had an ~ 2 0 of, ~0.9 S. Bezkorovainy (1965) found that he could retain acid glycoproteins from bovine blood serum, bovine colostrum acid whey, and bovine mature milk acid whey on a DEAE-cellulose column at pH 4.5 with a low I buffer. Subsequent elution resulted in predominantly h1-2 glycoprotein-like material in the first peak and M-1 glycoprotein-like material in the second peak. Chromatography on CM-cellulose columns was used to purify these fractions furtner. In the case of blood serum he was able to isolate orosomucoid from the M-1 fraction, and M-2 acid glycoprotein from the M-2 fraction. Colostrum acid whey contained appreciable amounts of orosomucoid and M-2 acid glycoprotein. The M-1 fraction containing orosomucoid also contained additional glycoproteins, at least one of which appears to be similar to one found in mature milk. Mature milk acid whey contained only traces of the M-2 blood serum acid glycoprotein in the first peak, and no detectable orosomucoid in the second peak, but there was a phosphoglycoprotein specific to milk in the second peak. This protein cm2V-'sec-' at pH 8.6 and -3.0 cm2V-%ec-' has a mobility of -7.1 X at pH 4.5 and a considerably lower carbohydrate fraction than orosomucoid. It has 0.44 gm phosphorus/100 gm protein, 13.6 % nitrogen, 4.0 % sialic acid, 2.4 % N-acetylhexosamine, and 3.1 % ' hexose. The amino acid composition and sedimentation behavior are different from that of K-casein. It gives a single boundary on sedimentation at pH 7.0 and pH 12.2 (phosphate) with an szo = 0.8 S. It is apparent that, despite discrepancies in the above work, there are present in mature milk several minor phosphoglycoproteins. The phosphoglycoprotein of Bezkorovainy (1965) is of special interest. However, when milk proteins arc treated with acid and heated, some of the phosphoglycoprotein and peptide material detected is probably the result of the rather rigorous treatment. It is important to establish unequivocally which of the minor phosphoglycoproteins occur naturally in milk. This will be possible only if the gentlest possible treatments are used. I t is also important to establish how many of these are lost in the various methods of

84

H. A. MCKENZIE

preparation of whole casein. Only 5 % of one such protein containing 2 % sialic acid would be needed to account for most of the difference in sialic acid content between whole acid casein and ammonium sulfate casein (the difference in NANA content is much greater than can be accounted for by the immunoglobulin content).

R . Methods of Electrophoretic Analysis of Caseins For nearly fifty years after Hammarsten’s early studies many workers Considered that casein was a pure protein. Linderstr@m-Langand Kodama (1925) showed by solubility studies that casein was indeed heterogeneous. Following the demonstration by Mellander (1939) that whole acid casein exhibited several peaks in moving-boundary electrophoresis, this method became the one of choice in casein heterogeneity studies. Unfortunately it became gradually apparent that the caseins interact with themselves and with one another. It was pointed out (Section IV) that we know now that simple interpretation of the patterns from transport experiments on complex systems is not possible. Many workers (including the reviewer) made the mistake of overinterpreting the patterns. Nevertheless it is apparent from the work of Warner (1944), Cherbuliez and Baudet (1950a), Kondo et al. (1950), Slatter and van Winkle (1952), and Tobias et al. (1952) that there are considerable interactions in the “a”-peak. Likewise the abnormal distribution of areas observed by Krecji et al. (1941, 1942) indicates interactions between “a,’- and “8”-protein fractions. McKenzie and Wake (1959b) made a moving-boundary electrophoretic study of acid casein, total milk protein, first cycle casein, second cycle casein-fractions P and S, a-casein, and p-casein (see Section V,C for preparation of these fractions). They showed that the splitting phenomenon observed for the a-peak by Warner (1944) did not occur for preparations of a-casein free of K-casein. They concluded that the splitting effect is due, at least in part, to the interaction of a- and 6-caseins. While they observed splitting in long runs on whole acid casein, no such effect was observed in samples of total milk protein or first cycle casein, indicating that the state of association of the a- and K-caseins may play a role. The nonenantiographic patterns, with a hypersharp ascending boundary, obtained for a-casein (free of K) indicate that a-casein may undergo a rapid association-dissociation reaction under the conditions of electrophoresis. McKenzie and Wake observed 80 yoof fast moving material and 2.0 % of slow moving material in moving-boundary electrophoresis of second-cycle casein fraction S at pH 7.0, and pointed out that if this distribution were assumed to be due to K- and @-casein,respectively, then there is disagreement between the K-casein content calculated in this way and that obtained from rennin action, etc. They suggested that this apparent discrepancy

MILK PROTEINS

85

arises because of complex reactions between the K- and a-caseins present (apart from questions of purity). There is no justification for ascribing either peak to either component alone. Thus the detection of “true” heterogeneity of casein fractions in electrophoresis is made considerably difficult by associationdissociation phenomena and interaction effects between the components. It became more and more apparent that, if reliable information on heterogeneity of casein were to be obtained, a method of resolution would have to be used in which the caseins were dissociated into their monomeric forms. A careful consideration of this problem was made by Wake and Baldwin (1961). They aimed to (i) disperse all the casein components into their monomeric form by carrying out the electrophoresis in 7 M urea solution, and (ii) take advantage of the high resolution offered by the Smithies (1959) starch gel method of electrophoresis. The most suitable buffer system they tried was the discontinuous Tris-citrate buffer system (pH 8.6) of Poulik (1957). They found, on electrophoresis of whole acid casein, over twenty bands, although only a few were major bands. Wake and Baldwin numbered the different bands according to their relative positions in the gel, the distance from the starting slot to an especially well-defined band being set a t 1-00. Typical patterns for acid casein and their enumeration are shown in Fig. V-la. The reproducibility of the band positions was found to be very satisfactory. The large number of bands observed came as a jolt to milk protein chemists, and the question arose immediately as to whether some of them a t least were artifacts. After elution of a number of zones from the gel they were re-run. The patterns obtained in each case were reproducible with respect to the number of bands observed and their mobilities. Patterns were obtained for several preparations of a-,@-, and K-caseins. A number of a-casein fractions were examined and found to have a major band identifiable with the major band in whole casein, but they also exhibited a large number of minor bands. Preparations of &casein showed considerable variation in heterogeneity, all giving one major band in common with the second most pronounced band in whole casein, but a number of the preparations showed other bands as well. A smeared zone was observed for K-casein with some sharp bands superimposed on the smeared band. This contribution of Wake and Baldwin is an important one and soon received much critical examination. Neelin el al. (1963) examined the effect of urea concentration in causing dissociation and of pH and buffer type on resolution. Buffers studied included acetate (pH 5.2-6.1), cacodylate (pH 6.2-7.2), phosphate (pH 6.0-7.4), Verona1 (pH 6.6-8.6), and borate (pH 10.41, all containing concentrated urea. Aggregation interfered near pH 5. Mobilities increased

86

H. A. MCKENZIE

with increasing pH, but above pH 7.0 the separation of major zones diminished, NeeIin et al. preferred pH 7.0-7.2 (cacodylate) for comparison of more mobile bands, but pH 8.2-8.4 (veronal) for slower moving bands. In the former pH range 5.5 M urea WM used, but in the latter range only 4.8 M urea was used in order to retain gel consistency. The problem of “smearing” and lack of complete reproducibility of K-casein behavior still remained. Subsequently Neelin (1964) suggested the use of small amounts of mercaptoethanol in the urea-gel buffer in the hope of dispersing the K-casein, since small amounts of S-S had been reported by others in K-casein (Waugh, 1958; Joll6s et al., 1962). Addition of mercaptoethanol overcame the smearing effect, improved the resolution in this region, and enabled genetic variants of K-casein to be detected (vide infra). Peterson (1963) found good resolution of ab- and fl-caseins by use of acrylamide gel electrophoresis with a 4.5 M urea, Tris-EDTA-boric acid

FIG.V-1. (a) Uretkstarch gel electrophoretic patterns of whole acid casein: the effect of protein concentration. Each band is numbered by the distance it has moved from the starting slot, relative to band 1.00. Protein concentration (in gm/liter) : (1) 15, (2) 8, (3) 5, (4) 2.5. (After Wake and Baldwin, 19Sl.) (b) Urea-mercaptoethanol starch-gel electrophoretic patterns of skim milk (Aschaffenburg and Thymann method) samples with the following variants: CY.,

(1) (2)

(3) (4) (5)

,-casein B BC B B B

@-casein A A AB A A

%-casein A A

AB

AB A

@-lactoglobulin AB AB B

AB AB

(by courtesy of Dr. R. Aschaffenburg). (c) Urea-polyacrylamide gel electrophoretic patterns (pH 9.1, 4.5 M urea) of the following @-caseinvariants: (1) A, (2) AB, (3) AC, (4) B, (5) BC, and (6) C. (After Thompson et d.,1964.)

(C)

FIG.V-1 (cont.). 87

88

H. A. MCKENZIE

(pH 9.0) mixture. This method has been applied very successfully by Thompson and his collaborators (e.g., Thompson et al., 1964) to the detection of genetic polymorphism in CY~J- and @-caseins(see Fig. V-lc for a typical example). Aschaff enburg (1964) has shown how the acrylamide method may be used directly for typing as,l-and 8-caseins by electrophoresis of whole milk samples. Investigators in several laboratories have adapted the acrylamide method for better resolution of K-caseins by the addition of mercaptoethanol. There is some difference of opinion regarding the extent to which prior removal of ammonium persulfate (used in the gel polymerization) is necessary. Aschaffenburg and Thymann (1965) have developed a method of 7 M urea-starch gel electrophoresis, using a Tris-EDTA-boric acid buffer (pH 8.9) and milk samples that have had prior treatment with mercaptoethanol. It is possible with this method to get good resolution for the caseins and some of the whey proteins (B-lactoglobulins B and C are not resolved, for example). Aschaffenburg and Thymann used thin gels on horizontal trays, and did not slice the gel prior to staining with amido black. Typical patterns are shown in Fig. V-lb. McKenzie and Murphy (1965) and Bell (1965) have found this method very useful. In one procedure, McKenzie and Murphy use a vertical electrophoresis apparatus, the milk samples are reduced overnight at 3’ using 1 yomercaptoethanol in 6.6 M urea, and, after electrophoresis in the cold room for 6 hours, the gels are sliced and stained with 0.1 % nigrosine for 5 minutes. Thompson (1964) has conducted a collaborative survey of the reproducibility of typing aB,l-and K-caseins by the various gel electrophoretic procedures used in a number of laboratories. On the whole the agreement between the various workers has been satisfying. Nevertheless the present procedures are empirical, and fundamental studies of resolution in zone electrophoretic procedures are needed to put electrophoretic analysis of caseins on a more rational basis.

C. Major Casein Fractions Following the resolution of whole casein into the a-,&, and y-peaks (in order of decreasing mobility at pH 7) in moving-boundary electrophoresis by Mellander (1939), attempts were intensified to fractionate casein into fractions with mobilities comparable to those of the a-,8-, and y-peaks. Among the first successful procedures was that of Warner (1944), who and “,5”-caseins based on the developed a procedure for separating ‘(a’’higher solubility of “@”-caseinat pH 4.4 and 2’. His method is outlined in Fig. V-2 and may be compared there with a number of other procedures. It is important to realize in considering Warner’s method that his acid casein was precipitated at pH 4.6 and 2 O , dissolved with enough sodium

MILK PROTEINS

89

hydroxide to attain a pH value of 6.5 and the solution extracted with ether, reprecipitated with 0.01 N HCl, all a t 2", and then washed and dried with alcohol and ether a t room temperature. The initial precipitation conditions would not normally bring down all the casein, and the subsequent treatment of the casein could have led to its modification. Subsequently Hipp et al. (1952) developed two fractionation procedures for acid casein. The first is based on differences in solubility of a-, p-, and y-caseins in 50 % alcohol in the presence of salt, as well as in water, with changes in pH and temperature. The second method depends on the differential solubility of the casein fractions in aqueous urea solutions near the isoelectric point. The first and second methods are outlined very briefly in Fig. V-2 for ease of comparison with other methods, but because of their importance in the development of procedures for the fractionation of casein they are given in greater detail in Figs. V-3 and V-4, respectively. Despite the fact that it was long known that much of the casein of skim milk existed as micelles incorporating calcium(II), it was not until the work of von Hippel and Waugh (1955) that use was made of such observations to separate casein from skim milk. A schematic outline of the preparation of casein by their method, and its partial fractionation (Waugh and von Hippel, 1956), is shown in Fig. V-5. Their method has been modified subsequently t o eliminate the oxalate method of removal of calcium. The more convenient citrate method is now used (Waugh, 1958). Evidence had accumulated that the protective colloid for the stabilization of the micelles in milk was a-casein, and that it was the component on which the enzyme rennin acted immediately (Cherbuliez and Baudet, 1950b). I n the course of their fractionation of skim milk, Waugh and von Hippel (1956) found evidence for the presence of a new component, which they designated K-casein. They considered this component, and not a-casein, t o be the protective colloid attacked by rennin. McKenzie and Wake (1959a) reasoned that these two points of view could be reconciled, if the a-casein samples examined prior to 1956 contained both an-casein and K-casein. They showed that K-casein is concentrated with a,-casein in fraction A during the alcohol fractionation procedure of Hipp et al. (1952). On the other hand, fraction B contains a,-casein essentially free of K-casein. The a-casein obtained in the urea fractionation procedure was found to consist of a mixture of as- and K-caseins. Thus only alcohol fraction B in these procedures was a suitable source for preparing a,-casein. McKenzie and Wake were able to show that those a-casein samples containing an-and K-caseins contained the protective colloid, whereas those containing only crn-caseinhad no protective properties. Following the work of Waugh's group, various attempts were made to fractionate casein into its major fractions by modifications of one or more

Fractionation of Casein

8

Warner (1944)

Hipp et al. (1952)

Waugh and von Hippel (1956)

(Solubility at p H 4, 2”) Acid casein, 2” (A to R.T.) (separate ‘a’,‘8’ by solubility at pH 4.2, 2”, high dilution) ‘a’: insoluble ‘8’: soluble

(Urea solubility) Acid casein, 25” (dissolve in 6.6 M urea, dilute to 4.6 M ) 1 . a . insol. (as, K) 1 7 p , ‘y ’ : sol. (dilute ‘ 8 , ~ ’fraction to 3.3 M urea ppt.; dilute super. + 1.7 M ) 8 : PPt. y: super.

(Ca++solubility) First cycle casein (make 0.25 M in CaC12,2’; warm to 37”) ppt. (blend, remove Ca) + second cycle-P (as, etc.) super. (remove Ca) -+ second cycle-S ( K , 8, etc.1

Cherbuliez and Baudet (195Oa) (separate ‘a’,‘0’ by solubility at pH 4.6, 4“; purify) ‘a’(remove y and u by solubility at pH 4.2, 50 gm/l (NH4)2SOa) : ppt. --+ la’ super. -+ u ‘0’ (remove ‘a’at pH 4.9, 2”; warm to 40”) PPt. B super. -+ y +

?.

(Alcohol solubility) Acid casein, 25“ (dissolve in NHdOAc-HOAc, pH 7; adjwt to pH 6.5) Fraction A: insol. -+ LY super. (to pH 5.7) Fraction B : insol. 4a. super. (cool to 2”) -+ “B”, “y”

McMeekin et al. (1959) E p p et al. (1961b) Acid casein, 30” [dissolve, adjust to pH 4.0, 99% insol.; dissolve ppt. at pH 7, adjust to pH 4.4. 4” (w)l

‘8, 7 ’ : super. [dissolve ppt., add CaCh to 0.2 M (231 ppt. + ffl (as,,) super. + LY2, as a a : ppt. pH 3.5 ( K ) a2:super. (A) ‘

aI : ppt.;

Groves et al. (1962) Acid casein, 25" [dissolve, adjust to pH 4.0, 2' ( W ) ] ppt.: reject super.: 8, y, etc. ppt.: 8, y, etc., at 26" [dissolve, chrometograph on DEAEcellulose column -+ 4 fractions. rechromatograph] Fraction 2 .--) y (S.G.) Fraction 3 4 B (S.G.)

'

Zittle and Custer (1963)

Acid casein, 20" [dissolve in 6.6 M urea. adjust t o p H 1.51 ~ u p a+ . K (S.G.) as:prepare by modified urea method (H) Manson (1965) Acid w i n , 25' [wash, pH 4.0 (McM); dissolve. pH 7.1; zonal density gradient electrophoresis (pH 7.1 and 2.3) --tag(S.G.) and

Schmidt and Pavens (1963)

Waugh et al. (1962)

Acid casein, R.T. First cycle casein [by 0.07 M Ca, 37"; suspend micelles in [dissolve p H 7.0, CaCll to 0.4 M (Z)] super. reject 0.01 M Ca, add K citrate p H 6.5, p p t . [suspend, remove Ca, dissolve in dialyze vs. NaOAc, add Ca to 0.17 M, 6.6 M urea, dilute to 4.6 M urea (HI]: 401 Ppt.: dissolve with K cit. p p t . [dissolve in 6.0 M urea, dilute to Reppt.: 0.07 M Ca, 5-7" 3.3 M (H)] ppt.: crude a-casein [dialyze K cit. + as;chromatograph on [column electrophoresis, pH 6.5, I 0.05, DEAEcellulose column, 4.5 M urea, 5 M urea ---t ms,l-casein (S.G.)] 13"+ two crrfractions (+others)] Payens and van Markwijk (1963): modification Hipp urea, Warner methods Manson (1961) + p-casein minor proteins (S.G.) First cycle casein [add CaClz to 0.4 M at p H 6-7.0 (Z)] Ribadeau-Dumas et d.(1964) ppt.: Ca ag-casein, etc. (+ 8, etc.) Acid casein, R.T. [add K cit. to remove Ca, dialyze, 0.085 M [dissolve pH 7, imidazole, 4.5 M urea; NaCl; repeat 4 a.] chromatograph on DEAE-cellulose column + 11 fractions] Fraction 7 --f p minor impurity (S.G.) Fraction 10 -+ as impurities (S.G.)

+

+ +

(S.G.)I

FIG.V-2. Key to symbols. (W) indicates principle of Warner (1944), (H) indicates principle of Hipp et a2. (1952), (Z) indicates principle of Waugh and von Hippel (1956), ( M c M ) indicates principle of McMeekin et al. (1959), (S.G.) indicates preparation checked by starch gel electrophoresis, and R.T. indicates room temperature.

92

H. A. MCKENZIE

Acid casein (dissolve at pH 7 with 0.1 N N&, 0.2 Y C & C O o ~ , and 50% alcohol)

(add N CH,COOH in 50% alcohol to pH 6.5) PreciDitate of fraction A (add 2 N C&COOH in 50% alcohol to pH 5.7)

(refractionate) %-Casein" (contains

I

K)

&pernatant (refractionate) +Casein

Precbitate of fraction C (dissolve to 1% at pH 7 with dilute NaOH and adjust to pH 4.5 at 2' with dilute HC1)

Supernatant

PreciDitate (discard)

(warm to 32')

P-casein

I

P-Casein

-supernatant containing 44%

y-casein and 44% of slower migrating component (refractionate) y-Casein

FIG.V-3. Alcohol fractionation of acid casein by the method of Hipp et al. (1952). (After McKcnsie and Wake, 1959a.)

93

MILK PROTEINS

Acid casein (dissolve to give 10% protein in 6 . 6 M urea, and dilute to 4.63 M urea)

-Precipitate

I

SUDeAatant

(refractionate)

I

(dilute to 3.3 M urea)

*%-casein"(contains K )

Supernatant

-Pre cipitaie (discard)

I(dilute to 1.7 M urea and and adjust to pH 4.7 with 0 . 1 N HC1)

(refractionate)

(refractionate)

of the basic methods of Hipp et al. (alcohol-urea), Warner (solubility at pH 4.0,4"), and Waugh (calcium) methods. These methods are outlined in Fig. V-2. Some indication is given of the major components in the fractions. It would seem that the al-,m-,and a3-fractions of McMeekin's group correspond roughIy to aE,l-,A-, and K-caseins, respectively. AII the fractions designated a.- are probably mainly a.,,-casein. Waugh et al. (1962) obtained the two fractions they called and a,,z-caseinfrom their cu.-casein. These seem now to be genetic variants of a.,l-casein (probably B and C). The procedures outlined in this section still form the basis of fractionation of most of the caseins. (Where significant departures from these basic methods occur, they are discussed in the respect,ivesection on the relevant casein component.) It is apparent that most of the procedures used

94

H. A. MCKENZIE

Skim milk

M Cac1, , centrifuge at 45,000 g and )'5

(0.06

Calcium ciseinate gel

Suaernataht (contains whey proteins)

(blend, remove Ca with oxalate, dialyze 2") Solution of firkt cycle casein (0.25 M CaCI, at 2", warm to 37",

and centrifuge a t 900 g and )'2

-Super'natant

Precioitate

I

(blend, remove Ca with oxalate, dialyze, 2")

I

(centrifuge at

9o,ooog, 2")

Solution of second cycle casein-fraction P

-Supernatant

Sediment (discard)

(remove Ca with oxalate, dialyze, 2') Second cycle casein-fraction S

FIG.V-5. Preparation of first cycle casein and second cycle casein-fractions P and S by the method of Waugh and von Hippel (1956). (After McKenzie and Wake, 1959a.)

involve rather harsh treatment of the casein. It is probably fortunate that most, if not all, of the caseins occur in milk as disordered-chain proteins and hence their native conformation may not be affected adversely by such treatment. Nevertheless the kinds of investigation now required

MILK PROTEINS

95

for the caseins, especially K-casein, are such that gentler methods of fractionation are sorely needed.

D . Formation and Stabilization of Casein Micelles 1. Introduction The central problem in understanding the biological function of milk proteins is the determination of the structure of the casein micelles and their interactions with the nonmicellar proteins and the inorganic and organic ions of milk. As well as being of great theoretical interest to protein chemists, any understanding gained of this problem is of great importance in problems arising out of the processing and storage of milk (see the reviews of Pyne, 1962; Rose, 1963, 1965; King, 1965). Despite great advances in knowledge of milk proteins we know very little a t present about the formation and structure of the micelles. The role of the inorganic and organic ions of milk in micelle stabilization, although of considerable importance, is very little understood mainly because there are tremendous difficulties in such investigations. I n discussing micelles, we shall first consider our present knowledge of the role of certain ions, and then the information gained about micelles from studies of synthetic mixtures of various caseins and calcium(II), taking note of important differences between the synthetic micelles and the micelles of milk. The immediate question that comes to mind is: what are the size and shape of the micelles of milk? Anoptral phase-contrast microscopy (King, 1960) and electron microscopy (Nitschmann, 1949; Hostettler and Imhof, 1951; Shimmin and Hill, 1964; Adachi, 1963; Knoop and Wortmann, 1960; Wortmann, 1965) of casein micelles indicate that they are roughly spherical in shape. Typical examples are shown in Fig. V-6. It is difficult to make exact estimates of micelle size because of the problems of “fixation.” However, t,he size distribution seems to be relatively narrow, ca. 40-300 mp. This corresponds very approximately to particle weights of lo6to 3 X lo9 and an estimate of 5-15 X 10l2 particles/ml milk. 2. Partition of Ions in Milk and the Role of Calcium Phosphate-Citrate

The ions of milk can exist as hydrated ions in the serum, or bound to proteins in this serum, e.g., the whey proteins or monomers and n-mers of the caseins (where n is small), or bound to the caseins in the niicelles, or bound to other ions. In the latter case, the resulting complex may be present as colloidal aggregates, which may be bound chemically or physically to the casein micelles (e.g., calcium(I1) and phosphate). It is extremely difficult if not impossible to work out an exact distribution for

96

H. A . MCKENZIE

FIQ.V-6. Electron micrograph of casein micelles fixed with osmium tetroxide and shadowed with platinum-palladium. Magnification, X50,OOO. (By courtesy of P. D. Shimmin and R. D. Hill.)

ions in so complex a system. However, a number of workers (e.g., Pyne) have devised ingenious ways of getting some idea of the ion distribution. All methods of doing this satisfactorily must aim at not disturbing the distribution that exists in the sample of milk being studied. The method of Davies and White (1960), in which a known volume of

MILK PROTEINS

97

milk is dialyzed against a relatively small volume of water at 20°, enables a good estimate to be made of the amount of various ions not in complexed form. In the case of calcium(II), analyses have usually been made by the murexide method of Smeets (1955; Tessier and Rose, 1958) or the ionexchange method of Christianson et al. (1954) and van Kreveld and van Minnen (1955). (Cf. Affsprung and Gehrke, 1956; Baker et al., 1954.) It seems from their results that the ion concentration of calcium(I1) in milk serum is ca. 2.6 mM/liter and of magnesium(I1) ca. 0.8 mM/liter. The various forms in which some of the ions of milk are present can be calculated approximately from the composition of milk serum and the dissociation constants of phosphoric, citric, and carbonic acids, after allowance has been made for binding of calcium(I1) and magnesium(I1) as citrate and phosphate complexes. Calculations (e.g., Smeets, 1955) based on these constants suggeet that ca. 55 % of the “soluble” calcium(I1) and magnesium(I1) is bound to citrate and ca. 10 % is hound to phosphate. The small amount of carbonic acid present occurs mainly as bicarbonate. I n considering the partition of the ions of milk, many workers have neglected the binding of both cations and anions by whey proteins and by monomers and small polymers of the caseins. This binding can be quite appreciable and more careful attention needs to be given to it; e.g., Zittle et al. (1957) have shown that Blactoglobulin can bind ca. 5 moles of calcium(I1) per (36,000) mole of B-lactoglobulin a t pH 6.8; Baker and Saroff (1965) have shown that @-lactoglobulincan bind ca. 1 mole of sodium(1) per mole (36,000)of Blactoglobulin at pH 6.9; Zittle et al. (1957) have found a maximum binding of 29 moles of calcium(I1) per lo6 gm casein a t pH 7.0;and Carr and Engelsted (1958) have shown that the maximum binding of sodium(1) by casein can attain a value of 18 moles/105 gm casein a t pH 7.5. Phosphate has been generally accepted as being the ion bound to calcium(I1) occurring in association with the calcium(I1) casein micelles. There has been considerable argument since the paper of van Slyke and Bosworth (1915) some fifty years ago as to the nature of the colloidal calcium phosphate aggregate formed, and whether or not it is bound in some way to the casein micelle. Resolution of this problem would be aided greatly if we knew the amount of calcium(I1) bound to the casein in the micelle. Casein contains a large number of ester phosphate groups (see the thorough review by Perlmann, 1955) and it now seems that all of these groups are monoesterophosphates (see review by Lindquist, 1963; and osterberg, 1966). Nevertheless it is not possible to calculate the extent to which the free acidic groups of the phosphate react with calcium(I1) (Schormueller and Fresenius, 1961), apart from the extent of their reaction with side-chain carboxyl groups.

98

13. A. MCKENZIE

Thus it becomes very difficult to make an indirect calculation of the amount of calcium (11) bound to the inorganic phosphate present. If we are to understand this problem of the nature of calcium phosphate aggregate properly, it is necessary to know soniething about the behavior of aqueous calcium phosphate systems. An excellent approach to this problem has been made by Roulet and Marier (1960, 1961) arid Boulet et al. (1962). This work has been summarized ably by Rose (1965) in his review of milk stability problems. Their results indicate that the calcium phosphate present in milk is utilikely to be a true di- or tricalcium phosphate, but is probably of the granular apatite type containing some citrate. This conclusion is based on the following observations : Freshly precipitated calcium phosphate can occur in two apatite forms: ( 2 ) a gelatinous form that is probably octacalcium phosphate, C a : P = 1.33:1, but is highly reactive and can exchange H+ or CaOH+ and thus form precipitates with Ca:P up to 1.66:1, (ii) a granular form that probably nucleates as dicalcium phosphate, but, is converted rapidly to a more alkaline form with Ca :P = 1.6:l. I t is unlikely that Cas(PO& ever forms from aqueous solutions of calcium(I1) and phosphate, and CaHP0 4is stable only within narrow limit,s of I , concentration and pH. (The magnesium(I1) of milk can play a role in the formation of the granular calcium(I1) salt.) Citrate tends to retard precipitation of both CaHP0 4 and the granular salt, but does not affect appreciably the type of precipitate formed. However, citrate can be incorporated into the granular salt to a varying degree. Pyne and McGann (1960) made a comparison of the composition of skim milk with that of a colloidal phosphate-free milk prepared so as to be approximately identical in all respects other than the colloidal phosphate content. The colloidal phosphate of milk is normally in equilibrium with serum approximately saturated with respect to calcium phosphate (Tessier and Rose, 1958). Pyne and McGann took advantage of the fact that the level of the colloidal phosphate in milk, whether it be t,hat originally present or not, tends to remain unaltered when the milk is dialyzed against a normal milk serum. They found that colloidal phosphate cotitents, 0-200 yo of that of the original milk (prepared by lowering or raising the pH value, respectively) , remained unaltered on dialysis against normal milk. Pyne and McGann were able to show from such experiments that the “calcium phosphate” colloidal aggregate is probably a calcium citrate-phosphate complex of the apatite type. The colloidal calcium phosphate is present in decreasing proportion in the casein niicelles as they decrease in size (Hostettler and Rychener, 1949; Ford et aZ., 1955). If the micelles are fractionated by centrifugation, depleted of calciuni(I1) by dialysis against a buffer (e.g., veronal), and then dialyzed against milk, the original micellar size reappears to some extent.

MILK PROTEINS

99

This means that the larger casein micelles in the original milk give rise to the larger recbonstituted micelles arid the smaller micelles to smaller ones. This phenomenon was named “size memory’’ by Choate et al. (1959) arid does not appear to depend on the fact that the colloidal calcium phosphate content of the original micelles is related to their size. I n fact, colloidal calcium phosphate does not enter the micelles recreated on dialysis against the original milk. The phenomenon of “size memory” is possihly more connected with the K-casein content of the micelles. Pyne and McGann (1960) preferred the idea of a chemical bond tietween the casein of the iiiicelle arid the calcium citrate-phosphate to mechanical inclusion. Pyrie (1962) found that r-casein was able to bind calcium phosphate to a higher degree than a- or p-casein. The effects of changes in temperature and pH are important in ;on distribution in milk arid have been discussed critically by Pyne (1962) in his excellent review (see also the recent work of Odagiri and Mickersoii, 1965). 3 . Foimation and Structure of Micelles

The idea t h a t one of the caseins acts as a protective colloid to stabilize the micelles of milk dates back to the work of Hanimarsten in 1872-1877. It received more definite formulation in the work of Linderstr@m-Lang (1929) and Holter (1932). In later work it was coricluded that a-casein was the protective colloid. The position changed radically when Waugh and von IIippel (1956) made the important discovery that the protective colloid is K-casein. It is not surprising that the attention of Waugh and his group was drawn to problems of the formation arid structure of casein micelles. In his initial work, Waugh (1958) found t ha t he could study the niicelle system converiiently by subjecting an aliquot of milk to high field centrifugation (140,000 9, 50 minutes) and determining subsequently the distribution of casein protein between “centrifugal” and “noncentrifugal” forms. He coricluded that ( i ) addition of calcium(I1) decreased progressively the amount of nonceritrifugal casein, (ii)dilution with physiological saline increased progressively the noncentrifugal casein, and (iii) addition of inorganic phosphate or other ions that sequester calciuni increased progressively the aniouri t of iioncentxifugal casein. Waugh proposed a model for casein micellcs iii which there were three a,-casein molecules oriented parallel to one another and placed symmetrically around a long rod-shaped K-casein molecule. Owing to the small size of the as-molecules, the K-casein would protrude partly from the casein bundle formed. The protruding part was assigned the protective colloid role. Within the micelle model, the four casein molecules were to be held together by hydro-

100

H. A. MCKENZIE

gen bonds and hydrophobic bonds. The K- and u8-casein molecules were considered to be so oriented that phosphate ester groups could be liiked through calcium. Waugh (1958) considered that the micelles of milk are in rapid equilibrium with their const,ituent components and complexes in solution. He concluded that micelles form only if a-and K-caseins are present simultaneously, and that their presence alone with calcium(I1) (4: 1,0.02 Ca(II),37”) is sufficient for micelle formation, although, if other caseins (e.g., @-casein) are present, they may be incorporated into the micelle. All the members of a series of divalent ions examined, except magnesium(II), formed micelles with us- and K-caseins under appropriate conditions: copper (0.03 M ) ; calcium, strontium, barium (0.03 M ) ; zinc, cadmium (0.007 M and 0.015 M ) ; manganese (0.015 M ) , cobalt, and nickel (each 0.03 M ) . Wake (1959b) examined the liberation by rennin (0.1 pg rennin N/ml) of the nonprotein” nitrogen (NPN) soluble in 0.73 M trichloroacetic acid, from various milk fractions a t pH 6.7 and 25”. He found that 1.0 yo nitrogen is released rapidly from whole (first cycle) casein, 3.4 % from from K-casein. No significant second cycle casein-fraction S, and 6.7 NPN release occurred in 20 minutes from milk fractions containing no K-casein. Ribadeau-Dumas and Alais (1961) and Malpress (1961) showed that rennin acts rapidly on casein micelles in mature milk to release some 65-85 yoof the total sialic acid. Rennin does not induce dispersion of the micelles in milk prior to their coalescence into paracasein curd (Hostettler and Imhof, 1951). The maximum stability of milk to heat is usually observed in the pH range 6.6-6.75, the minimum stability being a t slightly higher pH values. The maximum and minimum are dependent on the presence of @-lactoglobulin. It has been suggested that K-casein interacts with the 6-lactoglobulin during heating (Rose, 1961; Morr et d.,1962). Kannan and Jenness (1961) have shown that, in heated milk, @-lactoglobulincan react with the casein micelles sufficiently to delay clotting of the milk by rennin. The observations in the previous two paragraphs indicated that the K-casein must be near the surface of the micelle. Considering these observations and the electron microscopy work on casein micelles, discussed earlier, McKenzie (1963) suggested that the casein micelles are spherical, that the K-casein exists mainly near the surface of the micelle, and that the u- and 8-caseins are protected from the precipitation by calcium(I1) that occurs in the absence of K-casein. He rejected the earlier view of Waugh that “the constituents of the micelles are in equilibrium with their counterparts in solution.” McKenzie pointed out that in colloidal phenomena it is often difficult to differentiate clearly between rate and equilibrium effects. He suggested that the micellar system in milk can be described approxi-

MILK PROTEINS

101

mately as being diuturnal (i.e., the rate of change is not normally observable in the time of the experiment), but the calcium-cu.-K-casein system as caducous (i.e., the rate of change is more readily observable). This "transitory" nature can be illustrated by one of Waugh's experiments: if miceues of an-and K-caseins are formed in the presence of 0.03 M calcium(I1) a t 37" and the suspension is cooled t o 2", a precipitate forms slowly; but if the components are directly mixed a t 2" a precipitate forms more rapidly. Waugh's M.I.T. group recently reported a critical and careful study of the formation and structure of casein micelles. The essential findings of this work will be summarized briefly here. The reader is referred for experimental and other details to the extensive reports of Noble and Waugh (1965) and Waugh and Noble (1965). They studied casein interactions that take place prior to and during micelle formation, using mixtures of as-and K-caseins, or first cycle casein, or citrated skim milk (i.e., skim milk made 0.2 M in potassium citrate and then dialyzed vs. 0.07 M KCl adjusted to pH 7.2 with NaOH; "citrated skim milk" is called "solubilized skim milk" by Waugh). The techniques used involved analytical and preparative ultracentrifugation and, when calcium(I1) was present, measurements of supernatant protein at 37" after centrifugation for 1 minute at 400 g. an-Caseinand tr-casein were mixed, in ratios wrying from 4.5: 1 to 1 :1, at 2-6", without addition of calcium(I1). The ultracentrifuge patterns in phosphate buffer (pH 7.0, I 0.1) revealed only peaks characteristic of a*-and K-caseins. Pretreatment with urea, or taking the proteins to high pH values, did not alter the patterns. The same type of experiment was repeated at 20' with similar results, except when pretreatment took place. In the former experiments, there were observed a fast polydispene peak characteristic of K-casein with szO 2: 15 S and a slow peak characteristic of a.-caaein with h0= 3.9 S. Following pretreatment with 4 M urea some interaction was observed. When the an-casein and K-casein were mixed a t 37" in the ratio 3.5: 1, the ultracentrifuge pattern at 37" for the mixture in 0.07 M KC1 (adjusted to pH 7 with NaOH) was not the sum of the patterns of the individual proteins. Well-defined peaks with 520 = 3.4 S and 820 = 7.0 S were obtained. When the ratio was 1 :1, a single peak with 520 = 8.0 S was obtained. When there was pretreatment a t 37" with 4 M urea and the ultracentrifuge measurements were made a t 20" (pH 7, phosphate), patterns similar to those at 37" without pretreatment were obtained. These results indicate that an-and K-caseins do not interact in the absence of calcium(I1) at 2-6", interact at 20" only with pretreatment, and interact at 37" with or without pretreatment. The results are at variance with the earlier work of Waugh and von Hippel (1956). It is of interest to note that Pepper and Thompson (1963) mixed am-and K-caseins in the ratio 4: 1 at pH 7.0 and 25", both with and without dephosphorylation, and obtained

102

H. A. MCKENZIE

Molar concentration of CoCle (a 1

10

I

E

-e \

.i H

ac

0

5

b Q

m

0.01

0

a02

Molar concentration of CaCli ( b)

FIG.V-7.

See facing page.

103

MILK PROTEINS

6

LY,

,

,

I

I

1

I

,

0.05

0

1

,

I

0.I

I

I

1

I

0.2

Molar concentration of CaCI, (Cl

FIG.V-7. (a) Solubility of calcium a.-caseinate as a function of added CaCl2 for two solutions, both in standard KC1 buffer at 37". The o p a circles are for a solution with an initial a.-casein concentration of 10 mg/ml, the solid circ2es for a solution with an initial concentration of 5 mg/ml. The lines are calculated from Eq. 1 of Noble and Waugh (1965). (b) Supernatant protein resulting from single aliquot addition of calcium plotted as a function of CaC12 concentration for a series of cu.-K-casein mixtures in standard KC1 buffer at 37". Each solution contained initially 10 mg/ml of ascasein, but the initial K-casein concentration varied. The different K-casein concentrations are for 0.9 mg/ml, A for 0.8 mg/ml, for 0.7 represented as follows: 0 for 1 mg/ml, mg/ml, c]for 0.6 mg/ml, A for 0.5 mg/ml, and X for 0.0 mg/ml of K-casein, i.e., pure a,-casein. (c) Supernatant protein resulting from single aliquot addition of calcium plotted as a function of CaCh concentration for two aa-K-casein mixtures in standard KC1 buffer at 37". The main curve represents data for a solution initially containing 10 mg/ml of ascasein and 1 mg/ml of a-casein. The inset represents data for a solution initially containing 5 mg/ml of a.-casein and 2 mg/ml of K-casein. The designations of the segments of the curves in order of increasing CaCln concentration are the dip, the peak, and the pseudoplateau. (After Noble and Waugh, 1965.)

ultracentrifugal evidence of interaction. On the other hand, Swaisgood and Brunner (1962) found that spontaneous interaction of a.- and K-caseins does not occur at room temperature unless there has been prior treatment with urea or high pH, when complexing takes place in the ratio 4:l. Gamier et al. (1964b) obtained spontaneous interaction at 25' only when the weight ratio of the two proteins was unity. When Noble and Waugh mixed a,-casein with calcium(I1) at low concentration a t pH 7.0, no visible effect occurred. When the calcium(I1) concentration was increased to ca. 0.004 M (depending on protein concentration), precipitation occurred without micelle formation. The solubility relationships are shown in Fig. V-7s. Similar results were obtained for

104

H. A. MCKENZIE

mixtures of a,- and K-caseins when the calcium(I1) concentrations were low, but thereafter precipitation was progressively retarded by the presence of K-casein, to an extent corresponding to a weight ratio near 1 :1, just before micelle formation was initiated (see Fig. V-7b). Similar behavior was observed with first cycle casein and citrated skim milk. The addition of calcium(I1) in single aliquots to mixtures of an- and K-caseins to give concentrations between 0.007 and 0.02 M (depending on protein concentration) leads to increasing micelle formation and complete stabilization, if the initial weight ratio of a , : ~ was 110:1. A pronounced dip having descending and ascending limbs appeared in the plot of supernatant protein vs. calcium(I1) concentration (see Fig. V-7c). When a,:K was > 10: 1 the assay. centrifugate increased, but the amount of protein remaining as micelles exceeded greatly the sum of the initial K-casein and calcium an-caseinate solubilities. The occurrence of the dip is consistent with low weight ratio interaction products, since the dip decreases as the a,:K ratio decreases. When calcium(I1) was added in increments to solutions containing an-casein or mixtures of a.- and K-caseins at ratios < 10 :1, the descending limb of the dip appeared, as in the case of single aliquot addition, but leveled off at apparent an:^ stabiliziation ratios of 2-3:l (compared with 10-12 :1 for single aliquot addition). Binding of calcium(I1) was calculated from the displacement with respect to a.-casein concentration of plots of calcium a,-caseinate solubility vs. total calcium(I1). It was found that seven Ca(I1) ions were bound per a,-casein molecule just prior to precipitation, with a further four during formation of the precipitate. Waugh and Noble concluded that: (i) Prior to micelle formation at 37”, free calcium a.-caseinate and a low weight ratio calcium a,-K-caseinate interaction product are present. (ii) Formation of stable micelles requires a minimum level of calcium(I1) greater than that required to precipitate the free a,-casein present. (iii) The weight fraction of K-casein in a micelle decreases with increasing micelle size (Sullivan et al., 1959). (iv) Precipitates formed from mixtures of an- and K-caseins with calcium(I1) in the region of the “dip” (Fig. V-7) are calcium an-caseinate. Those formed in the region of the “peak” and beyond (i.e., at Ca(I1) concentrations sufficient for micelle formation) are different from the calcium a,-caseinate precipitates in containing small amounts of K- casein, in being nonadherent, and in compacting to give white opaque pellets. (v) Micelles have a size distribution that depends strongly on the initial a,:K ratio, the absolute protein concentration, and the calcium(I1) concen-

MILK PROTEINS

105

tration on single aliquot addition. The apparent final states of the systems are dependent on the path. (vi) Stabilization in the region of the “peak” can be achieved a t all a,:K ratios up to a t least 10:1. ( v i i ) The size distributions of micelles can be altered rapidly (minutes) by the addition of K-casein and probably over some hours by the addition of calcium(I1). (viii) A micelle distribution, once formed, is relatively stable to dilution with a buffer containing an appropriate concentration of calcium(I1). ( i z ) Micelles are highly solvated. (z) Although micelles require only the presence of as-and K-caseins, they can incorporate @-casein. Thus Waugh was led to modify some of his earlier ideas on the structure of the micelle. He now considers the path dependencies observed by Noble and Waugh to indicate that the micelle system is not an equilibrium system (under their experimental conditions), and the micelles to be spherical and to consist in simplest form of calcium a,-caseinate covered by a uniform coating of low weight ratio calcium am-K-caseinate. (These conclusions may be compared with the recent discussion by Payens, 1966.) The recent observations of Waugh and his group on micelles are of considerable interest and importance. They should be studied closely, with a view to designing future experiments on micelle structure and stability. It will be necessary to reexamine the problem of the interaction of casein components in the absence of calcium(II), in view of the conflicting results on complex formation of aa- and K-caseins. It will also be necessary to extend this work to determine what happens when calcium citrate-phosphate is present.

E . a,-Caseins 1 . Genetic Variants of a&asein

Aschaffenburg’s (1961) demonstration of the occurrence of genetic variants of @-caseinwas the first concrete evidence of genetic variants in the components of casein. Following this work, Thompson el al. (1962) obtained evidence of the occurrence of three genetic variants in a,-casein. Subsequently Kiddy et al. (1964) examined casein samples prepared from individual milk samples of a large number of cows, using the urea-starch gel method of electrophoresis described by Wake and Baldwin (1961). Their results indicated that cr.,l-casein (for nomenclature see Section 111) occurs in various forms, the occurrence of which is controlled by three allelic autosomal genes with no dominance. Each allele is responsible for pro-

106

H, A. MCKENZIE

duction of one of the three forms of a,,l-caseiri, which are designated a,,1-A1 aaS1-B,LY,,~-C in order of decreasing mobility in urea-starch gel at pH 8.6. Milk from individual cows may contain any one or two of these variants. The Philadelphia group examined milk samples from 1378 cows and obtained the following distribution of a.,l-casein types: Aynhire, 98 B; Brown Swiss, 192 B, 11 BC; Guernsey 188 B, 180 BC, 32 C; Holstein 2 A, 81 AB, 5 AC, 410 B, 44 BC; and Jersey 44 B! 21 BC, 2 C. Some cross-breeds were tested, giving 67 B and 1 BC. It is to be emphasized that the data do not necessarily reflect the frequencies occurring in random samples of the breeds, since sire groups and breeds were often selected in an attempt to find certain variants. The reasons why the B allele is so common and the A so rare are not apparent. 2. Isolation of Genetic Variants of aBgl-Casein

General methods of isolation of a,-caseins have been discussed (Section V,C). Thompson and Kiddy (1964) have developed a procedure for isolation of a,.,-caseins A, B and C from the niilk of animah homozygous for the particular variant desired. They precipitated acid casein at pH 4.6 and 25O, washed the precipitate, dissolved it in 6.6 M urea, and then diluted it to 3.3 M (cf. procedures of Hipp et al., 1952, in Section V,C) to precipitate the “a”-casein. The a,-casein was precipitated from the “a”-casein by 0.4 M calcium chloride. This was followed by an alcoholammonium acetate fractionation and then chromatography on a DEAEcellulose column (3.3 M urea, 0.01 M imidazole-HC1). The cw,,rcaseins obtained showed only a single band on gel electrophoresis. It is not possible by this method to separate heterozygous mixtures of the variants.

5. Molecular Size of as-Caseins

Svedberg et al. (1930) first estimated the molecular weight of the major sedimenting component in acid casein to be 75,000-100,000. Burk and Greenberg (1930) found from osmotic pressure measurements that the molecular weight of whole casein in 6.6 M urea was 33,600. Hipp et al. (1952) showed that urea can disperse casein aggregates. D’Yachenko and Vlodovets (1952) showed that disaggregation could be effected also by extremes of acidity and alkalinity. Perlmann (1954) suggest’ed a minimum molecular weight of 31,000 for “al’-caseiii on the basis of its phosphorus content. A careful study was made by Halwer (1954) of the variation in light scattering due to association-dissociation of “a1’-and @caseins, prepared by the method of Warner (1944) (see Section V,C). Halwer found and &caseins was strongly dependent that the polymerization of both ((a”on electrolyte concentration at pH 7, and that their light scattering be-

MILK PROTEINS

107

havior resembled that of denatured proteins. (However, a t high pH values there was much less polymerization and much less dependence on electrolyt,e concentration.) The tendency t.oward polymerization increased with increasing temperature. The polymerization of “ a”-casein was rapid (equilibrium in ca. 1 minute) whereas that of &casein was slower (ca. 45 minutes). The polymerization of “cr”-casein was readily reversible, that of p-casein not so easily reversible. It is apparent that the polymerization is endothermic in both cases. This taken along with the ionic strength dependence makes it likely that hydrophobic binding is involved. McKenzie and Wake (1959~)determined the molecular weight of a,-casein piepared from the alcohol fraction €3 of Hipp et a,?. (see Section V,C). They attempted to dissociate the a,-casein into its monomeric form by using as solvent (a) glycine buffer pH 11.0, Z0.20, (b) 6 M urea, 0.05 M phosphate buffer pH 7.3. It was found that the concentration dependence of sedimentation a t pH 11.0 was high, making difficult accurate extrapolation of the sedimentation coefficient to zero protein concentration SO^^,^). The value of so20,w was estimated as 1.61 f 0.03 S. The diffusion coefficient (D) was not very dependent on concentration, and a value of 5.9 Fick units was estimated for Do20,w. Combining these figures with a value of 0 of 0.728 ml/gm, the molecular weight was computed to be 25,000 f 1OOO. The concentration dependence of s and D in the 6 M urea solvent was low, and extrapolated values of sozo,w= 1.33 S and D = 4.33 Fick units were obtained. Assuming the same 6 value, the molecular weight was estimated to be 27,600 f 1OOO. No correction was made for selective solvation, and this may be appreciable. An estimate of molecular weight a t pH 12.0 ( I 0.19, phosphate) was made by the Archibald sedimentation method and a value of 25,500 f 1000 was obtained. Dreizen el al. (1962) made a light scattering study of the a,-casein preparation of Waugh et al. (1962). This preparation seems to be a.,l-casein BC, free of contaminants by urea-starch gel electrophoresis. They found differences in the state of association of protein dissolved directly into pH 12.0 buffer and of protein dissolved a t pH 7 and dialyzed against pH 12.0 buffer. In the former case they estimated a mdecular weight of 27,000 f 1000 and in the latter case 27,300 f 1000. They found a number of unpredictable polymerizations occurring, especially in dialyses a t low ionic strengths (<0.3) and low protein concentrations. Dreizen et al., in comparing their results with those of McKenzie and Wake, stated that the a,casein of McKenzie and Wake had been prepared by calcium and alcohol precipitation and was found by Wake and Baldwin (1961) to contain several components. This statement is incorrect : the “ a”-casein samples examined by Wake and Baldwin were prepared by other methods and were not the preparations of McKenzie and Wake. The latter’s preparation was

108

H. A. MCKENZIE

prepared by alcohol fractionation from fraction B in the method of Hipp et al. This fraction was chosen on account of its relative freedom from K-casein. Errors in the values obtained at pH 12.0 arise mainly from the high concentration dependence of the parameters being measured for the highly charged protein, which makes extrapolation to zero concentration uncertain. It is difficult also to compare the values for the pH 12 solvent with the values for the 6 M urea-pH 7 solvent, since no allowance was made for selective solvation in the latter case. Schmidt and Payens (1963) examined their preparation of a,,l-casein BC (see Section V,C) by the Archibald sedimentation method and obtained a value of 16,500 for the molecular weight a t pH 12.2 ( I = 0.5, glycine buffer) arid 2.5". This value seems to be in erior, especially in view of the minimum molecular weight of 30,000 expected from Manson's finding of one N-terminal arginine per 30,000gm a.-casein. More recentIy Payens and Schmidt (1965, 1966) made a study of the association of cw..l-caseins B and C . They found that at pH 6.4 each of these caseins undergoes rapid endothermic polymerization, and that the particle weight of the polymerizing unit for cr,,rcasein B is 87,500and for rYs.rcasein C is 113,000 f 3000. They examined the boundary spreading of each of these caseins during sedimentation in the light of the theories of Gilbert and Fujita (see Section IV). They considered that there were consecutive polymers up to the pentamer of the polymerizing unit in each case, and computed sedimentation patterns, without allowing for diffusional spreading or the concentration dependence of sedimentation. The experimental patterns were unimodal in every case with a trailing edge apparent a t the lower concentrations. Payem and Schmidt are aware of the dangers of ascribing this unimodality to stepwise formation of polymers. The majority of their computed curves display a single maximum but a t high concentration there is some evidence of bimodality in the computed curves, which would be detected only with difficulty in the present case. They made apparent weight average molecular weight measurements by the Archibald sedimentation method. Using a modification of the Steiner (1952) procedure, they calculated stepwise association constants at 2", go, and 14". They found AH and A S to be positive and the free enthalpy was decreased by a constant amount of -3.2 kcal/mole at each association step up to the pentamer. The earlier studies discussed above indicate that the polymerization of the monomer of "a"-casein (molecular weight ca. 26,000) is rapid. Thus it is difficult to accept Payens and Schmidt's conclusion that the polymerizing unit is 87,500 and 113,000 for aE,l-caseinsB and C , respectively. One wonders if their measurements were not carried out a t a sufficiently low concentration to detect the dissociation of these units into the monomer units of 26,000 (molecular weight),

MILK PROTEINS

109

4 . Chemical Composition of a,-Caseins The amino acid composition of a1-casein (approximate to a.,*-casein

BC) was determined by Hipp et al. (1961a) and is included in Table V-2. Their results may be compared with those of Ho and Waugh (1965a,b) for their a,-casein, which is probably a,,l-casein BC. Gordon et al. (1965) determined the amino acid cornposition of a,,l-caseins A, B and C, while de Koning and van Rooijen (1965) determined the composition of the B and C variants. Examination of Table V-2 indicates that there is close

agreement between the analyses made by the Philadelphia and Dutch groups. However, there are some discrepancies between the results obtained by Ho and Waugh for a,,l-BC and those of the Dutch group. The results from the Philadelphia group indicate that the C variant has one more glycine residue per molecule than B, and that the glycine might be replaced by a glutamic acid residue. The differences in composition between the A and the other variants are so great that it is difficult to account for them. The work of Kalan et aE. (1964) indicates that the amino-terminal amino acid is arginine in all three variants in accord with the earlier work of Manson (1961) on pooled aR,l-casein. The result's of the action of carboxypeptidase A indicate a leucyltryptophan carboxyl-terminal sequence for all three variants. A molecular weight of ca. 31,000 was calculated from the carboxypeptidase data, assuming a single polypeptide chain. Some ten years ago Perlmann (1955) reviewed her work on the phosphate bonds in casein. She classified the phosphorus bond in a-casein as 40 % mixed N-P-0 diesters, 40 % 0-P monoesters and 20 % O-pyrophosphate diesters. Thoai et al. (1954) reported 33 % diesteis and 53 % monoesters in a-casein. Subsequently evidence accumulated indicating the presence of only one type of phosphate ester in casein, probably the O-monophosphate ester (Hofman, 1958; Kalan and Telka, 1959a,b; Anderson and Kelley, 1959). Osterberg (1959) characterized the phosphate esters by their pH titration curves. Since the phosphate groups ( < 8 %) make only a small contribution to the total number of titratable groups, osterberg degraded the protein enzymatically to a small peptide and then titrated its phosphate groups. In this way he found the three phosphate groups of a peptide from whole casein to be of the O-monophosphate ester type. Subsequently usterberg (1960, 1961) studied a peptide from "a"-casein, in which seven phosphate groups were concentrated , and again demonstrated the presence of O-monoesters. Further enzymatic hydrolysis of the peptide liberated O-phosphorylserine ( h e r b e r g , 1964). From a series of such studies h e r b e r g concluded that 70 % of the phosphorus in a,,l-casein is present in the sequence shown in Fig. V-8, and this

110

H. A. MCKENZIE

TABLE V-2 Amino Acid Composition of Bovine as-Caseins: Comparison of Various Workers’ Results (Residues per monomer M.W. 27,000) Variant: Workersa: GlY Ala Ser Thr pro Val Ileu Leu Phe TYr CYSP Met ASP Glu NHs Arg His LYS TotalN (%) Phosphorus (yo)

a1

a6.1

- BC

(~6.1

-A

(HBG) (HW) (KR)

(GBT)

10.2 10.3 9.4 4.9 13.7 11.9 12.3 21.5 9.9 12.3 2.3 0 5.9 13.9 44.1 15.4 8.6 5.8 17.8 14.7 1.0

10.3 9.5 17.2 6.5 19.9 11.5 13.1 16.7 7.2 11.7 2.7

8.5 9.6 14.9 6.8 18.2 12.3 10.6 17.8 8.3 10.6 2.8 0.4

4.4 15.4 38.3 27.0 5.8 4.6 15.8 14.1 0.85

10.3 9.9 14.8 5.8 18.9 12.6 12.2 18.8 8.7 10.8 3.3 0 5.4 17.1 44.6 33.6 6.7 5.5 15.3 15.01 1.12

as.1

(KR)

10.1 10.3 15.8 5.4 18.4 12.4 12.1 19.0 8.9 10.8 3.2 0 0 5.7 5.4 16.2 16.9 44.9 44.4 26.1 30.5 6.0 6.6 6.0 5.7 17.4 15.4 15.10* 14.37 1.01* 1.12

-B

aa.1

-c

(GBT)

(KR)

(GBT)

10.1 10.2 16.3 5.7 19.2 12.6 12.4 19.2 9.1 10.9 2.5 0 5.4 17.1 43.8 29.4 6.8 5.8 16.0 15.34* 1.01,

11.2 10.3 15.7 5.5 18.9 12.7 12.3 19.0 8.9 10.8

11.1 10.2 16.6 5.8 19.2 12.8 12.5 19.3 9.2 11.0 2.6 0 5.4 17.2 42.9 28.0 6.8 5.8 16.0 15.40* 1.01*

3.3 0

5.3 16.6 43.3 31.2 6.6 5.7 15.3 14.3 1.12

HBG = Hipp, Basch, and Gordon (1961a); HW = Ho and Waugh (1965a); KR = de Koning and van Rooijen (1965); GBT = Gordon, Basch, and Thompson (1965). Asterisks refer to data of Thompson and Pepper (1964b). (I

P1

I

Asp-(SerP, Ile) -(Asp, ThrP, Gly, GI%)- (SerP, Glu) -GluP2 Ilea, G14, Asp, Va&, Lys, Pro)-

Ala-SerP-SerP-(SerP,,

t

t

P4

p3 GluNH, - Glu- (Ala, Met)-Asp-Glu- (GluNh, Met) -1le

- Lys

FIG.V-8. Partial structural formula of the tryptic -casein phosphopeptide. The lysine residues are marked in italics and the phosphorylated amino acid residues in boldface t g p e . The arrows indicate major points of peptic hydrolysis. The peptides shown between these arrows are designated by increasing P numbers. T w o amide groups are not indicated; residues containing these groups are assigned as Asp or Glu. (After bierberg, 1966.)

MILK PROTEINS

111

constitutes one tenth of the molecule (molecular weight ca. 30,000). Osterberg (1966) has reviewed his careful work and compared the phosphorylated sequences of casein with other phosphoproteins. Ho and Waugh (1965a,b) have studied the role of organic phosphate groups in the interaction of bovine caseins with metal ions. Manson (1965) has studied the role of the organic phosphate groups of caseins in the binding of basic dyes. 6. Conjormation of a-Casein

Evidence has accumulated over many years that the caseins resemble denatured proteins in their behavior in solution. This property is no doubt advantageous in its role as a food for the newborn, because a disordered configuration should make it particularly susceptible to digestion. Optical rotation measurements have indicated that a-casein probably has a disordered configuration (e.g., Hewitt, 1927; Cohen and Szent-Gyorgyi, 1957; McKenzie et al., 1955). The high content of proline may be sufficient to prevent helix formation (see also Kirchmeier, 1962). Despite such observations the idea has persisted in the literature that a-casein is a rod-like structure with an axial ratio of ca. 15. Recent light scattering studies by Krescheck (1965) indicate that a-casein exists as a random coil in solution a t pH 6.5.

F. K-Caseins 1. Isolation of K-Casein In their original work Waugh and von Hippel (1956) detected the presence of the new component K-casein, and obtained fractions rich in this component, but did not isolate it. Soon after this work McKenzie and Wake (1959a) examined a number of the fractions of casein for the presence or absence of the K-casein (as discussed in Section V,C) and Wake (1959a) succeeded in isolating K-casein, containing only a few percent of impurities, from second cycle casein-fraction S. In this procedure partial removal of @-caseinwas effected by procedures based on the alcohol fractionation method of Hipp et al. (1952). The pH value of the supernatant was adjusted to 5.7; enriched K-casein was precipitated, and redissolved at pH 7. The K-casein was precipitated a t 2" and pH 4.6 by use of the procedure of Warner (1944) to separate it from the more soluble @casein. Subsequently McKenzie and Wake (1961) considered that it should be possible to prepare K-casein from acid casein. They developed the improved procedure shown schematically in Fig. V-9. This method has been widely employed. Use is made of features of the alcohol fractionation method of Hipp et al., once the second cycle casein-fraction S is obtained. If this procedure is to be

112

H . A. MCKENZIE

Swaisgood-Brunner (1962)

McKensie-Vi7ake (1961)

Cheeseman (1962)

Acid casein Dissolve pH 7.0, cool, 2"; make 0.37 M in Ca++, warm 35"; reject ppt.; remove Ca from super., warm to 25", add Na2S0, (250 gm/l), dissolve ppt., adjust pH to 7.2; fractionate with 50 % EtOH-2 M NHaOAc, dissolve p p t . 6 M urea, dialyze vs. 0.005 M NaCl; refractionate with EtOH, dissolve, dialyze

Acid casein Dissolve 6.6 M urea, prepare 'a-K'fraction by urea met.hod of Hipp et al., dissolve a t pH 7, remove residual Ca with oxalate, cool, 4", make 3.3 M in urea, adjust pH to 4.9, remove p p t . of as, dialyze super., make 0.2 M in Ca++, warm 37". 1 hr; super. -)K

Acid casein Dissolve 6.6 M urea, prepare 'WK' by urea method, dissolve 6.6 M urea, make 12% (w/w) TCA; p p t . : as, etc.; super.: adjust to pH 7, dialyze, concentrate; alter to pH 8, make 0.25 M Ca++, 3"; super. + K fraction; adjust pH 11 and then to pH 4.4+ PPt. K

Zittle-Custer (1963)

Hill (1963)

Acid casein (frozen block) Dissolve 6.6 M urea, adjust to pH 1.5; super. -+ K-fraction; p p t . with (NH&S04 (1 M ) ; dissolve pH 7.5, dialyze

Acid casein Dissolve pH 8 (50 gm/l), cool, 3"; make 0.13M Ca++, warm 35': dialyze wiper. vs. H20, concentrate, make 0.3 M Ca++; dialyze super. VY. pH 6.2.5 (acetate) buffer; chromatograph on DEAEcellulose columns

FIG V-9. Methods for the preparation of K-casein.

used successfully, careful attention must be given to detail, especially the purity of chemicals (e.g., ammonium acetate; see Beeby and Nitschmann, 1963). The observations by Hipp et al. (1952), that acid caseii is soluble in concentrated urea solution and that the glycomacropeptide, prepared by action of rennin on casein, is soluble in 12 % (w/w) trichloroacetic acid, led Swaisgood and Brunner (1962) to separate K-casein in such media. Their preparation is outlined scheniatically in Fig. V-9. Zittle and Custer (1963) precipitated ag and &caseins from urea solutions of acid casein by addition of sulfuric acid to pH 1.3. Ammonium sulfate was added to the supernatant to precipitate K-casein, as shown in Fig. V-9. Zittle (1965) has shown tthat K-casein prepared by the ureasulfuric acid method has 4 times the protease activity of the whole acid

MILK PROTEINS

113

casein from which it is prepared. He found that sulfuric acid had certain specific effects, compared with other acids, in the casein fractionation. Cheeseman (1962) preferred to prepare K-casein from as-^ rather than second cycle casein fractions. He prepared as-K-casein from acid casein by the urea fractionation procedure of Hipp et al. (1952), and removed a,-casein by addition of calcium(II), as shown in Fig. V-9. Hill (1963) developed a method for preparation of K-casein from acid casein, based on chromatography on a DEAE-cellulose column (shown in Fig. V-9). Many of the above methods for the preparation of g-casein use acid casein as starting material, and involve some harsh procedures. All the preparations appear to exhibit some degree of heterogeneity when examined by sensitive methods. It will be seen in subsequent parts of this section that rather critical work is now needed on K-casein; c.g., to determine whether K-casein in milk contains SH or S-S groups. For such investigations to proceed satisfactorily very gentle methods for fractionation of K-casein will be needed. Even precipitation at low pH should be avoided if possible. During the last three years McKenzie and Murphy (1965) have endeavored to develop such procedures. They used ammonium sulfate casein and first cycle casein as starting materials. Attempts were made to separate K-casein by column electrophoresis on a variety of supporting media. Considerable purification has been achieved, but there still remains some 3-5 % of material not readily removed from the K-casein by gentle procedures It is not yet certain whether at least part of this material is a phosphoglycoprotein associated with K-casein. The discovery of genetic variants of K-casein has given new impetus to the search for new methods of isolation. 2. Genetic Variants of K-Ca.sein

It was pointed out (Section IV,C) that the smeared pattern, given by K-casein in gel electrophoresis even in the presence of 7 M urea, has been a matter of some concern to those investigating the heterogeneity of the caseins. It was pointed out that there was some evidence that the K-casein fraction, alone among the casein fractions, appeared to contain cystine and possibly cysteine. Thus attempts were made about the same time in several laboratories to determine if polydispersity due to intermolecular S-S linkages were mainly responsible for the smeared pattern. Mackinlay and Wake (1964) treated K-casein, prepared by the method of McKenzie and Wake (1961), with sodium sulfite to break any S-S bonds that might be present. They then examined the product by the urea-starch gel electrophoretic method of Wake and Baldwin (1961). The S-sulfo-K-casein showed two msjor sharp bands and three minor sharp bands on gel electro-

114

H. A. MCKENZIE

phoresis. Likewise Neelin (1964) found two major sharp bands in K-casein, which had been prepared by the method of Zittle and Custer (1963) from pooled milk and had been treated with mercaptoethanol to reduce S-S bonds. On the other hand, reduced K-casein preparations from milk of individua,lcows showed the two main bands or either one of them. When the two bands occurred together, they did not stain with equal intensity. Neelin suggested that the bands may represent genetically controlled variants of K-casein. At about the same time, Woychik (1964) isolated K-casein from whole casein of milk of individual cows, using the urea-sulfuric acid method of Zittle and Custer (1963) followed by purification by a procedure based on that of McKenzie and Wake (1961). He examined the K-casein samples by the polyacrylamide gel electrophoretic method of Peterson (1963) after the samples had been reduced with mercaptoethanol for 3 hours. Patterns revealed the presence of three major bands, several intermediate bands, and several minor bands. He found that the major bands tended to occur singly or in pairs, and suggested that the presence or absence of these bands was under genetic control. Subsequently Woychik (1965) pointed out that several other groups of workers found only two major bands occurring in K-casein from individual cows. Thus he was prompted to repeat his earlier work; as a result of his later work, it is apparent that one of his earlier K-casein types is artifactual. Schniidt (1964) made an independent study of K-casein types, using whole acid casein and also K-casein piepared from individual cows by the method of McKenzie and Wake (1961). He reduced the samples in a solution containing 2-mercaptoethanol followed by urea-starch gel electrophoresis. As a result of this study of preparations from 48 Friesian and 22 MRIJ cows, he concluded that there are only two individual K-casein variants in these two important Dutch breeds of dairy cow. Mackinlay and Wake (1965b) applied the method of Schmidt (1964) to the examination of casein samples from milk of individual cows. They observed only two major bands in the patterns of reduced acid casein from 34 cows, and found rather wide variation in the mobilities of each of these bands, relative to the band designated 1.00 (see Section V,B). The two major bands may be designated A and B in order of decreasing mobility. Some sampIes had A and B in amounts that stained equally intensely in the gel. Some samples contained A without evidence of material staining in the B position. Others had a single major band in the B position together with a small amount of material migrating in the A position. Some samples had a major band in the A position and a minor band in the B position. All samples had minor b n d s migrating more rapidly than A. A feature of all the patterns is the lack of bands due to y-casein, which are

115

MILK PROTEINS

normally detectable in the 0.34 and 0.41 positions for whole casein run in the absence of reductant. Typical patterns are shown in Fig. V-10 I t seemed possible to Mackinlay and Wake that the minor band in the B position could be due to y-casein. Accordingly, a sample of each of the four phenotypes (assuming each of the major bands is genetically controlled) was carried through a reduction and alkylation procedure (see

FIQ.V-10. Urea mercaptoethanol-starch gel electrophoretic patterns of whole acid caseins from milk samples of individual cows: (a) B and A bands in equal amounts, (b) major B band, minor band in A position, (c) major A band, no band in B position, (d) major A band, no band in B position, (e) major A band, no band in B position, (f) B and A bands in equal amounts, (g) major A band, no band in B position, and (h) major A band, no band in B position. (After Mackinlay and Wake, 1965b.)

Section V,F). Samples of each of the S-carboxylmethyl derivatives (SCMcasein) were examined by gel electrophoresis in the absence of mercaptoethanol, both before and after the action of rennin, in order to determine which of the bands might be ascribed to K-casein and y-casein. Samples of K-casein, which under reducing conditions gave the B the minor band in the A position or the A band only, gave SCM-K-casein derivatives that had major bands at the 0.52 and 0.59 positions, respectively. Samples, whose patterns had the B and A bands in amounts that stained equally,

+

FIQ.V-11. Urea-starch gel electrophoretic patterns at pH 8.6 of (a): (1) ScM-K-CaSein (10 gm/l) from pooled milk, (2)K-casein (10 gm/l) from pooled milk; and of (b): (3) pooled SCM-K-CaSein (10 gm/l), (4)SCM-K-casein B (10 gm/l), (5) SCM-K-casein A (10 gm/l), (6) pooled SCAM-K-casein (10 gm/l), (7) pooled para-SCM-K-casein (20 gm/l), and (8) pooled para-SCAM-K-casein (20 gm/l). (After Mackinlay and Wake, 1965b.) 116

117

MILK PROTEINS

gave rise to SCM derivatives whose patterns gave the 0.52 and 0.59 bands in equal intensity. A sample that gave the A band a minor band in the B position gave a derivative in which there werP two bands barely resolved from one another in the 0.59 and 0.61 positions. When these patterns were compared with those of the same samples after rennin treatment, it was concluded that the material giving rise to the minor band in the B position is not affected by rennin, is possibly a y-casein, and is not the Same protein that gives rise to the B band. All the K-casein variants gave only one paraSCM-ti-casein on rennin action. Hence any amino acid replacements responsible for the variants must reside in the glycomacropeptide portion of the molecule. The major bands of SCM-ti-casein occur in the 0.59 and 0.52 positions; the two ti-casein variants giving rise to these bands are designated the A and B variants, respectively. I n patterns for the A variant there is no band in the 0.52 position. However, in patterns of the B variant there is a minor band in the 0.59 position. It can be seen, in Fig. V-11; that the patterns for the two variants are very similar, both having a single major band and several minor bands, the mobility of the bands for the SCM-Kcasein A all being slightly higher than the corresponding bands for the SCM-ti-casein B. The patterns of the two variants are consistent with the idea that the components giving the minor bands arise from the major components. This will be discussed further (Section V,G,3). First we shall discuss the preparation and some properties of reduced K-casein derivatives.

+

3. Intermolecular Disulfide Bonds in K-Casein and Th,eir Cleavage

Mackinlay and Wake (1964) prepared the S-sulfo derivative of K-casein (S03-~-casein)by reaction of the protein with sodium sulfite in the presence of phenylmercuric hydroxide. The overall reactions are :

+

+

R'--S--SR* SO&*- -iRLS-SOsRCSRLSceH~,-Hg+ + RP--S--Hg-CsHs

+

Their derivative showed five sharp bands in urea-starch gel electrophoresis. All these bands disappeared following rennin action. Rennin caused the S03-ti-caseinto precipitate in the same way as for ti-casein, and some 24 yo of the total nitrogen became soluble in 2 Yo (w/w) trichloroacetic acid. Zone electrophoretic analysis of the resulting para-SOrKcasein indicated the presence of two bands, one major and one minor, migrating toward the cathode at alkaline pH. The S03-~-casein was shown by Mackinlay and Wake to stabilize cw.,l-casein in the presence of calcium(I1) to form micelles. An examination of the sedimentation properties of S03-ti-casein in a range of dissociating solvents indicated that it

118

H. A. MCKENZIE

dissociated readily from a polymer with an S Z O ,= ~ 9.1 S at neutral pH to a form having an szo.w = 1.5 S in 20 yo dimethylformamide and 25 % (v/v) dioxane. This behavior may be contrasted with the behavior of K-casein, which cannot be dissociated very effectively in these solvents. It also lends support to the idea that at least part of the size heterogeneity of K-mein arises out of intermolecular disulfide bonding. It is evident that, depending on the sulfur atom to which the sulfonyl group becomes attached, two structural isomers are possible if the disulfide bond is intramolecular. Thus Mackinlay and Wake (1965a) considered it desirable to prepare a different derivative of K-casein, in which the same blocking group is attached to both sides of the disulfide bond. This was done by reducing K-casein with mercaptoethanol and then alkylating the resultant sulfhydryl groups with iodoacetate to give S-carboxymethyl (SCM) deritatives. A typical starch gel electrophoretic pattern at pH 6, for SCM-K-CaSein prepared from pooled milk, is shown in Fig. V-lla. The series of sharp bands obtained has already been described briefly in connection with genetic variants of K-casein. Their relative mobilities are indicated in Fig. V-11. The two bands, 0.52 and 0.59, are the major bands, as we have seen previously. The 0.66 band appears to consist of at least two components on the basis of other work carried out by Mackinlay and Wake. The 0.74 band appears to be a doublet. The poorly resolved, rapidly moving bands have mobilities very close to that of @casein. The presence of sniall amounts of &casein contaminants in most preparations of SCM-K-casein makes difficult the characterization of these bands. Mackinlay and Wake deduced that at least three of the bands in the 0.794.84 region are SCM-Kcasein bands on the basis of experiments involving the action of rennin. They concluded, from comparison of patterns of SCM-acid casein and SCM-K-casein, that only bands due to K-casein are altered by the reduction and alkylation procedure. This is in accordance with Waugh's finding that the K-casein fraction is the only casein fraction containing S-S bonds. Mackinlay and Wake (1965a) developed a procedure for the fractionation of SCM-K-casein by chromatography on DEAE-cellulose columns at 20" and pH 7.0 (KHzP04, K*HP04,KOH, H3BOs) in the presence of 20 % (v/v) rlimethylformamide as dissociating agent. A typical elution pattern and the variation in composition of eluting buffers are given in Fig. V-12. Results for the urea-starch gel electrophoretic examination and carbohydrate and phosphorus analyses of the fractions are shown in Table V-3. It can be seen that none of the fractions exhibits a single band in starch gel electrophoresis. Thus it is not possible to obtain either of the major components (0.52 or 0.59) free of contaminants by this fractionation procedure. The total phosphorus is distributed fairly evenly among the

119

MILK PROTEINS I.OL

0.9a0 -

0.7-

a6 5

t

P a3a2 -

ai 0-r 0 Fraction number

FIG.V-12. Elution pattern for fractionation of SCM-K-CaSein by chromatography on DEAE-cellulose (flow rate, 30 ml/hr; individual fractions, 7.5 ml). The buffer composition was changed at the points indicated by the arrows,as follows: (I) 0.0184 M (total) phosphate, (2) 0.0207 M phosphate, (3)0.0230M phosphate, (4) 0.046 M phosphate, and (5) 0.023 M phosphate, 0.15 M NaC1.

fractions, but most of the carbohydrate content is restricted to the minor fractions containing material giving rise to faster moving bands in electrophoresis. The two major fractions contain virtually no carbohydrate. It was found that fractions F2, Fs, and Fs stabilized casein micelles equally well. Thus it was concluded that carbohydrate is not essential to the micelle-stabilizing capacity of SCM-K-casein. TABLE V-3

Composition of SCM-dmein Fractionsa ~

~~

Residues/106 gm protein Electrophoretic bands

Fraction

Impurities 0.52, minor 0.59 0.59, minor 0.66 0.59, minor 0.66 0.59, 0.66 0.74, minor 0.66,O. 79

F1

Fz

Faa F3 Fab

F,

F.5

0.71?-0.84

~~

a

After Mackinlay and Wake (1965a).

Hexosamine

NANA

-

-

-

-

1.1

0.3 0.4

0.3

4.8

Hexose

2.2

-

2.6

5.8 14.9

-

1.1 3.8 9.9

0.6 1.5

6.3 16.8

P

-

5.8

-

5.2

6.8

120

H. A. MCKENZIE

Mackinlay and Wake (1965a) also carried out a similar fractionation on SCM-acid casein and obtained comparable resolution to that obtained by Ribadeau-Dumas et al. (1964), using DEAE-cellulose and elution buffers containing urea. Neither group of workers achieved satisfactory separation of p- and K-caseins. Subsequently Mackinlay and Wake (1965b) fractionated the individual SCM-K-caseins (the 0.52 and 0.59 components). They failed to obtain either variant completely free of other material.

4. Molecular Size of K-Casein Soon after Waugh and von Hippel (1956) made their important discovery of the occurrence of h-easein, it was isolated in relatively pure form by Wake (1959a). He found that his preparation gave a major, fast moving peak, with a very small, slow moving peak, in sedimentation velocity experiments at pH 7.0 ( I 0.1, phosphate). The fast moving peak had an s20 of ca. 13 S at a protein concentration of 10 gm/liter, but the peak showed evidence of appreciable boundary spreading on the leading edge. The sedimentation pattern was quite different at pH 12 (I0.19, phosphate) and consisted of a single symmetrical peak of szo = 1.0 S at a protein concentration of 10 gm/liter. McKenzie and Wake (1959~)measured the apparent molecular weight of this preparation by the Archibald sedimentation method. It was assumed that the protein was dissociated completely into the monomeric form and that the partial specific volume was 0.73 ml/gm. The apparent molecular weight values were extrapolated to zero concentration to give a value of 26,000 f 3000. While McKenzie realized that, if this protein contained S-S linkages, there would be considerable cleavage of these linkages under such alkaline conditions, he did not check on this. I t will now be seen that subsequent studies by other workers have revealed that polymerization of K-casein is through S-S linkages and these are broken at pH 12. Mackinlay and Wake (196513) determined the disulfide content of K-casein, by a method similar to that used for other proteins by Cecil and Wake (1962). They found 5.8 f 0.3 moles of S-S groups/106gm K-casein, a i d less than 0.3 mole of available SH groups/106 gm protein. This value is similar to the value of 1.4 gm cystine/100 gm protein, reported by Jollks et al. (1962). Swaisgood and Brunner (1962) had pointed out earlier that the S - S linkages of K-casein would probably be destroyed at pH 12. Mackinlay and Wake (1965b) investigated this possibility and found that 4 of the S-S content of K-casein was lost after 24 hours at pH 12.0,23 yowas lost in 24 hours at lo", and, at 20", 11 % was lost in 1 hour, 29 yoin 8 hours, and 82 % in 21 hours. They also made a study of the sedimentation behavior

MILK PROTEINS

121

at pH 12.0 and concluded that complete dissociation was effected only after all the S-S linkages had been broken. Swaisgood et al. (1964) determined the weight average molecular weight 11guanidine hydrochloride and obof K-casein in 67 % acetic acid and 5.0 1 tained a value of 125,000. They concluded from data, obtained by the Archibald method, that the smallest particle present had a molecular weight of 56,000 under these conditions. A value of 28,000 was obtained for the molecular weight of mercaptoethanol-reduced K-casein. Mackinlay and Wake (196513) measured the apparent weight average molecular weight of SCM-K-casein in 20 % dimethylformamide at pH 7.0, using the sedimentation equilibrium method. They obtained extrapolated values of the 19,000 for the weight average molecular weight and 18,700 for the Z-average molecular weight. A value of 19,300 was obtained for for the weight average molecular weight at pH 12 ( I 0.19, phosphate) and a value of 20,400 in 50 % acetic acid. They concluded that the molecular weight of SCM-K-casein is 19,500 f 600, assuming 5 = 0.73 ml/gm. (These values are some 25 % lower than the earlier value of McKenzie and Wake for K-casein, presumably because the S-S had not been completely cleaved under the conditions used.) 6. Chemical Properties of K-Casein

The amino acid analysis of arcasein was determined by Hipp et al. (1961a). It will be recalled that this preparation appears to consist largely of K-casein. Its amino acid composition, nitrogen content, phosphorus content, and carbohydrate composition are given in Table V-4 for comparison with results for K-casein, given in the same table. The latter results include those of Jollhs et al. (1962) and of Swaisgood and Brunner (1962), along with those of Woychik and Kalan (1965) for K-casein, S-cyanoethylK-casein, and S-carboxamidomethyl-K-casein. The amino acid analyses of the various %-caseinpreparations are fairly close to one another. It will be noted that the cystine contents have not been corrected for destruction during hydrolysis. Thus they are low compared with the values reported by Mackinlay and Wake (196513) and Jollks et a1. (1962). Amino acid analysis of only one of the known individual variants of K-casein appears to have been made. However, analysis of the B variant has been made and is given in Table V-4. It should be stressed that the sialic acid, nitrogen, and phosphorus concentrations of K-casein preparations reported in the literature have a wide variation: the sialic acid 0.79-2.50 gm/100 gm, the nitrogen 13.6-15.4 gm/lOO gm and the phosphorus 0.22-0.35 gm/100 gm. The carbohydrate contents of K-casein, prepared by a given procedure, can vary widely from milk sample to milk sample. Marier et al. (1963)

122

H. A. MCKENZIE

found that the sialic acid content of whole acid casein preparations could vary from 0.26 % to 0.59 %. They concluded that this result indicates wide variation in K-casein content. However, this variation could be due to differences in K-casein content and/or variation in the proportion of carbohydrate-rich components. Ribadeau-Dumas and Veaux (1964) TABLE V-4 Amino Acid Composition of Bovine K-Casein: Comparison of Various Workers’ Results (Residues per monomer M.W. 20,000) Type : Workersa :

GlY Ala Ser Thr Pro Val Ileu Leu Phe TY~ Try CYSP Met

A B ~

Glu

NHa

Arg His Lys Total N Phosphorus NANA Hexosamine Hexose

ruJ

K

K

K

K-B

(HBG)

(JA)

(SB)

(HHB)

(ICW)

3.4 12.4 10.8 7.1 17.8 8.8 9.8 9.8 4.8 10.8 1.8 1.1 1.5 11.4 24.5 24.7 5.2 1.9 9.3 14.6 0 35 1.21

3.5 12.1 11.6 11.1 15.2 8.7 9.4 9.3 4.9 8.2 1 .o 1.2 1.3 11.0 23.6 13.0 4.6 2.2 7.9 0.22 2.4 1.2 1.4

3.3 12.2 9.6 11.3 19.0 10.8 10.8 9.3 4.7 8.4 1.o 2.3 11.6 26.9 22.8 4.5 3.0 8.9 15.3 0.22 1.4

4.0 9.7 9.9 10.5 19.5 6.4 7.3 7.0 4.3 7.6

2.6 13.4 11.0 12.1 17.5 10.2 11.5 8.7 4.2 8.8

1.6 13.1 26.5 25.8 3.8 2.8 1.2 14.39

1.9 1.9 11.0 24.9 5.0 3.0 9.1 -

-

0.75

-

-

-

1.37 0.61 0.92

-

-

-

-

HBG = Hipp, Basch, and Gordon (1961a); JA = Jollbs, Alais, and Jollbs (1962); SB = Swaisgood and Brunner (1962); HHB = Huang, Hennebeny, and Baker (1964); KW = Kalan and Woychik (1965).

found that the sialic acid content of acid casein derived from bovine colostrum contained 0.85 yo sialic acid and liberated 2.95 % nitrogen soluble in 12 % (w/w) trichloroacetic acid. They assumed this to be due to the variation in K-casein content. Malpress and Hytten (1964) found in their careful studies of human casein a direct relationship between the sialic acid in the isolated whole

MILK PROTEINS

123

acid casein and the noncasein sialic acid in the milk. Both decrease during lactation. Alais and Jollhs (1962) noted the release of a glycopeptide, relatively rich in sialic acid, by the action of rennin on human acid casein. Mackinlay and Wake (1965b) made a study of the starch gel electrophoretic patterns of SCM-acid casein prepared from cow colostral milk. They found little variation in the K-casein content of the whole casein at the first and fourth days, but noted variations in the proportion of bands containing carbohydrate. Hill and Hanson (1963) studied the effect of preparative conditions on the composition of K-casein. They found that the ratio of sialic acid to cystine may be changed considerably as a result of alcohol fractionation, although the actual sialic acid contents of chromatographically and alcoholfractionated K-casein are similar. Their results were interpreted in terms of the ideas of Beeby (1963) that K-casein is a “complex” of three proteins of similar molecular weight, one having all the sialic acid, the second all the cystine, and the third containing neither sialic acid nor cystine. All the above results lead to the conclusion that the carbohydrate content of K-casein fractions of a single milk sample varies according to the preparative procedure. The variation in sialic acid content of whole acid catkin preparations is due to variation in the K-casein content and/or the sialic acid content of the K-casein. This indicates that the sialic acid content of whole casein is not a reliable index of its K-casein content. The absence of cysteine from acid casein was reported first by Kassell and Brand (1938). Waugh (1961) concluded that whole casein contained cystine (see also Yoshino et al., 1962) and that all the cystine is in the K-casein fraction. The work on Ic-casein in this section supports this view. However, Beeby (1964) made the important observation that freshly prepared second cycle casein-fraction S contains cysteine only, and that the sulfhydryl groups present are “masked” by calcium(I1) so that they do not react with the usual SH reagents. He also showed that sulfhydryl groups could be detected readily under dissociating conditions at pH 9, after the last traces of calcium(I1) had been removed from the K-casein fraction by addition of oxalate or EDTA. However, he was unable to detect cysteine in his preparations of K-casein made by the method of McKenzie and Wake (1961). Thus he concluded that the cysteine had been oxidized to cystine during the fractionation of the K-casein. Subsequently R. D. Hill (1964), working in the same laboratory, was able to detect cysteine in whole acid casein preparations only after they had been digested with pronase. The question of whether casein in milk contains cysteine has not yet been resolved, although it is being investigated in several laboratories including that of the reviewer.

124

H. A. MCKENZIE

G. Rennin and Its Action on %-Casein

I . Introduction

As a result of his investigations of the action of rennin on casein, Hammarsten asked the following question nearly a century ago, “Is there a substance in the milk and in the casein solutions able to dissolve casein calcium phosphate which will be destroyed by the rennet, and is cheese only the casein calcium phosphate made insoluble by destruction of this compound?” Over fifty years later Linderstrprm-Lang (1929) and Holter (1932) developed the theory that casein micelles are stabilized by a protective colloid, and that the protective properties of the colloid are destroyed when the relevant casein component. is modified slightly by the enzyme. The main features of this theory are still essentially valid. Nitschmann and his collaborators concluded, as a result of extensive studies, that there is an enzymatic primary phase (optimal over the pH range 3.5-5.5) in which the protective colloid properties arc destroyed by the enzyme, and that this is followed by a secondary stage of coagulation of casein (see Nitschm a h and Lehmann, 1947; Nitschmann and Zahler, 1950; Nitschmann and Varin, 1951; Mattenheimer et al., 1952). Later Alais et al. (1953) concluded that there is also a tertiary stage in which all casein components are slowly hydrolyzed as a result of the proteolytic activity of rennin. Cherbuliel; and Baudet (1950b) concluded that “a”-casein is the protective colloid. We have discussed previously how we know now that this “a”-casein contained K-casein and that it is, in fact, the K-casein that is the protective colloid. Alais el al. (1953) demonstrated that, during the action of rennin on ( 1ar 11- casein, “nonprotein nitrogen” (NPN) is released and part of this NPN is soluble in 12 yotrichloroacetic acid and part is soluble in 2 yotrichloroacetic acid (pH 4.7). Alais (1956) and Nitschmann et al. (1957) ’ trichloroacetic acid, and concluded that ca. 1.5 % NPN, soluble in 12 % 4 % NPN, soluble in 2 % trichloroacetic acid, are split rapidly from whole casein by rennin at 25’. They concluded also that the NPN soluble in 12 yotrichloroacetic acid is primarily a glycomacropeptidewith a molecular weight of 6000-8000 (see also Nitschmann and Henzi, 1959). Wake (1959b) showed that 1 % NPN, soluble in 12 % trichloroacetic acid, was released rapidly from his preparations of whole casein, 3.4 % from second cycle casein-fraction S and 6.7 % from K-casein. None was split from the other milk proteins under similar conditions. The work of Tsugo and Yamauchi (1960) seems to indicate that, during the primary phase of rennin action, the only change brought about is the loss of the macropeptides, the micelles remaining intact. The secondary

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phase comprises many different steps of aggregation, coagulation, and syneresis. Scott ‘Blair and Oosthuizen (1961, 1962) followed the viscosity changes of K-casein during rennin action. The viscosity decreased linearly in the initial stages; then it decreased nonlinearly, passed through a minimum and began to increase; then aggregation occurred, Newton’s law being obeyed. Finally, the viscosity rose without Newton’s law being obeyed. They concluded that the decrease in viscosity was a viscoelectric effect and that the increase in viscosity was connected with aggregation. The syneresis of the casein gel has been discussed by many workers and several stepwise mechanisms for it have been proposed. The processes of rennin action on casein are complex, and attempts to describe each of the stages by single reaction kinetics have not been very successful. Furthermore, modern kinetic criteria have not generally been applied in this work. The work up to 1963 on the action of rennin on casein has been reviewed thoroughly by Lindquist, (1963) in his review on casein. Thus no attempt is made t o describe this work in detail here, but some of its relevant features are given as a basis of our discussion of recent work. Before discussing this work we shall consider briefly some aspects of the chemistry of rennin. 2. Properties of Rennin

While the use of “rennet” preparations from calf stomachs to clot milk has long been known, it is only comparatively recently that the crystallization of the enzyme responsible, namely, rennin, has been achieved. The first procedure for crystallization is due to Berridge (1945) and was modified by Berridge and Woodward (1953). Numerous workers reported difficulty in the crystallization of rennin by these methods and Berridge (1955) made a number of pertinent suggestions on how to overcome these difficulties. More recently Foltmann has made a thorough study of the isolation and properties of rennin and its precursor, prorennin. Foltmann (1959a)b)prepared extracts of calf stomach, and clarified and activated them in one process with aluminum sulfate and disodium hydrogen phosphate. This was followed by two precipitations with sodium chloride. The crystals were soluble in distilled water to the extent of a t least 80 gm/liter, but only sparingly soluble (0.2 gm/liter) in 1 M sodium chloride at pH 5.5. He found the pH range of optimum stability of solutions of his preparation to be 5.5-6.0, the enzyme being unstable around pH 3.5 and above pH 6.5. Soon afterward Foltmann (1960a) carried out a chromatographic fractionation of solutions of his rennin crystals. The solution was applied to a column of DEAE-cellulose equilibrated previously with 0.2 M phosphate

126

H. A. MCKENZIE

buffer, pH 5.7. Following elution of a small amount of inactive material (3 yo),three active fractions were eluted with 0.225-0.25 M phosphate buffer. The first fraction eluted, C, was about 20 yo of the total and had an activity of 55-60 RU, the second fraction, B, ca. 55 %, had an activity of 100 RU, and the final fraction, A, ca. 22 had an activity of 125 RU. (The enzyme activity is expressed in rennin units, RU, according to the definition of Berridge, 1945.) Foltmann (1958) also prepared prorennin from dried calf stomachs, achieving partial purification by salt fractionation. Later this preparation was purified further by fractionation on DEAEcellulose columns (0.10 M PO,, pH 5.8; elution with 0.10-0.25 M PO4). The prorennin, eluted in a single peak at 0.20 M PO4, was rechromatographed at pH 5.7-5.8 (Foltmann, 1960b). Part of the material was activated at pH 2.1 for 15 minutes and 1.5 hours, before rechromatography. These experiments showed the presence of A and B rennins in equal amounts after the short activation period, but the presence of A, B, and C rennins after the longer period at pH 2.1. The purified prorennin had a single N-terminal amino acid residue: namely, alanine (1 gram mole residue/40,000-50,000 gm prorennin). The sedimentation pattern had a single peak, with an so20,w= 3.5 5, and showed increasing 8'20 ,wwith increasing concentration, indicating polymerization. Later Foltmann (1962) rechromatographed this prorennin preparation on a DEAE-cellulose column (equilibrated at pH 5.9, 0.05 M POr),eluting with a linear gradient of phosphate buffer. He obtained an inactive peak followed by a second highly asymmetrical peak containing the prorennin activity. The latter material was divided into two fractions, designated A and B, and rechromatographed. Following a series of activation experiments, Foltmann concluded that rennin A corresponds to prorennin A, and rennin B to prorennin B. The origin and nature of fraction C are obscure. Foltmann concluded that the activation of prorennin takes place by splitting peptides off the N-terminal end of the proenzyme. Rand and Ernstrom (1964) found the sodium chloride concentration and pH value to have a considerable effect on the rate of activation of prorennin as well as the find yield of activity. Greatest yields of stable activity were obtained by activation at pH 5.0 in the presence of 1.5-2.0 M sodium chloride. Activation was more rapid at lower pH values, but the activity was not stable. They found that there was a limited initial nonautocatalytic activation at pH 5.0 until the activation became predominantly autocatalytic. Djurtoft et d. (1964) studied the sedimentat.ion behavior of prorennin and rennin at pH 5.8. In each case a single, slightly asymmetrical peak

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was observed. The sedimentation coefficient increased with increasing concentration, indicating polymerization. An estimate was made of s ~ = 3.5 ~ S~and,3.2 ~S for the proenzyme and the enzyme, respectively. Unfortunately these measurements were not made down to very low concentration, and the extrapolation is uncertain in the reviewer’s opinion. Combining these sozo,rr values with a DOZo,w= 8.5 Fick units and a B = 0.73 ml/gni, a molecular weight of 34,000was calculated for rennin. Although the concentration dependence of s, and the single peak obtained, suggest a rapidly polymerizing system, no systematic investigation has been made of this. Gel filtration experiments were carried out, with Sephadex G-100. Unfortunately these were not of the type recommended in Section IV, thus no general conclusions can be made at present regarding the nature of the polymerization reactions. Foltmann (1964a) has carried out amino acid analyses of prorennin, rennin, and the peptides released on activation of the proenzyme. Foltmann and Hartley (1966) have determined the amino acid sequences of peptides, accounting for 99 of the ca. 270 residues in rennin. The sequence around three disulfide bridges has been determined, one of which forms a small internal loop of five residues. Six different arginine sequences were found, whereas amino acid analyses allowed only five. This suggests heterogeneity in the sequence of crystalline rennin. A sequence of 11 residues at the cmboxyl terminus has been indicated. Foltmann (1964b) has compared the proteolytic activity of different rennin preparations with that of pepsin. Using denatured hemoglobin as substrate, all the preparations had an optimum activity at pH 3.5. The ratio of proteolytic activity to milk clotting activity was different for all the fractions. Using the B-chain of oxidized insulin as substrate, the products of digestion by rennin and pepsin were analyzed by two-dimensional paper electrophoresis and chromatography (Bang-Jensen et al., 1964). The fractions from digestion by the various rennin preparations were all the same, but different from those obtained by pepsin digest.ion. Some, but not all, of the sites of attack by rennin and pepsin were similar. Fish (1957) reported that rennin had a lower proteolytic specificity than pepsin. However, the Danish group considers that the sequence Leu-Val No. 11-12, which is one of the main targets of pepsin attack, is attacked only very slowly by rennin. On the other hand, the Leu-Val sequence No. 17-18 is hydrolyzed rapidly by rennin and is almost unaffected by pepsin. A difference occurred, not only in the site of attack by the two enzymes but also in the effect of the pH value on activity. The enzymes behaved differently also toward ribonuclease. It was inactivated by pepsin but not by rennin.

128

H. A. MCKENZIE

3. The Mode qf Action of Rennin on K-Casein

Jollbs et a2. (1961) have made a study of the glycopeptide obtained after the action of rennin on whole acid casein and on K-casein from the milk of the cow, sheep, and goat. They found the peptide part of the glycopeptide to differ for the various species. The C-terminal amino acids were alanine and valine for the cow, and glutamic acid and valine for the sheep and goat. Arginine and aromatic and sulfur-containing amino acids were absent. The presence of phosphoserinewas detected. The molecular weight of the glycopeptides was 600CL8000 and there was nearly 30% carbohydrate content. The glycopeptide originates from the C-terminal portion of the r-casein, since the same amino acids are C-terminal for both the K-casein and the glycomacropeptide. Alais and Jollbs (1961) showed that the carbohydrate moiety of the glycopeptide contains bound phosphate, galactose, galactosamine, and sialic acid, which is released by neuraminidase and occupies a terminal position. Jollbs et al. (1962) found a new C-terminal phenylalanine residue in K-casein (para-K-casein) following rennin action. However, no new N-terminal residue was detected. Hence it wafi suggested that rennin might be splitting an ester linkage rather than a peptide linkage. Jollb et al. (1963) found that lithium borohydride acted on K-casein to produce two fragments of amino acid composition similar to those of the para-K-casein and the glycomacropeptide. This result was taken to lend support to the idea that an ester linkage was being split by rennin. However, Crestfield et al. (1963) have shown that sodium borohydride can cleave a peptide bond in ribonuclease. Thus a peptide bond rather than an ester linkage could be cleaved in K-casein. Gamier el a2. (1962) measured the uptake of protons during the action of rennin on r-casein. They interpreted their results to indicate that an ester linkage is the site of rennin attack. Delfour el al. (1965) have now found that there is an N-terminal methionine residue in the glycopeptide, which had previously escaped detection because it is broken down rapidly during the hydrolysis conditions employed in N-terminal residue determinations. Thus it is not necessary to postulate an ester linkage as the site of rennin action on K-casein (cf. the work of Humme, 1965, on low molecular weight, peptide substrates for rennin and the work of Dennis and Wake, 1965, on the action of proteolytic enzymes on K-casein) . Jollbs (1965) has found that the N-terminal sequence of the glycopeptide contains three lysine residues, the tentative sequence being : Met-Ala-IleuPro-Pro-Lys-Lys-(Glu-Aspp)-Lys-Thr.The carbohydrates seem to be situated in the COOH-terminal end: the sialic acid is terminal and galactosamine is linked to the peptide part.

MILK PROTEINS

129

Malpress and Seid-Akhaven (1965) have found quantitative differences in the glycopeptides of human casein and bovine casein, alt,hough glycine, leucine, and methionine may be absent in the human glycopeptide. The human glycopeptide contains f ucose and glucosamine, which are absent in the bovine glycopeptide. It also contains galactose and galactosamine. Fractionation of tryptic digests of the glycopeptides indicated the possibility of -Lys-Pro- linkage. The existence of O-glycosidic linkages to threonine and serine was inferred in the human glycopeptide. Hill and Laing (1965a) have concluded that photo-oxidation of K-casein in the presence of methylene blue causes the loss of the ability of K-casein to be split and clotted by rennin. They conclude that both these effects are caused by alteration of histidine residues and that the latter may be part of the site of rennin action on K-casein (see also Hill and Laing, 196513). We have seen that the peptide material released by action of rennin on K-casein is heterogeneous with respect to its solubility in trichloroacetic acid. Some 25 of the total nitrogen of K-casein is rendered soluble in 2 yo trichloroacetic acid and some 12 % ’ in 12 % ’ trichloroacetic acid. The latter fraction, the glycopeptide. contains most of the carbohydrate of K-casein. The question arose as to whether rennin split more than one peptide from each K-casein molecule, or whetsherthe heterogeneity of the peptide material released arose from heterogeneity of the K-casein. We have seen how Mackinlay and Wake (1964, 1965a,b) showed that SCM-K-casein is heterogeneous, and that the various fractions differ in carbohydrate content ’ but possess similar ability to stabilize casein micelles. They from 0 to 10 % carried out experiments on the action of rennin on their SCM-K-casein fractions and showed that the heterogeneity of the soluble material released by rennin action is a function of the heterogeneity of the SCM-Kcasein. Since rennin acted equally well on all the fractions irrespective of whether they contained carbohydrate or not, Mackinlay and Wake have rejected an ester linkage as the site of action of rennin on K-casein. It will be recalled that, in Fig. V-10, uren-starch gel electrophoretic patterns of SCM-K-casein and SCAM-K-casein are shown for pooled material and for the individual variants. Also shown are patterns for these derivatives after the action of rennin. These patterns indicate that the major and minor bands of the para-K-casein derivatives are formed from the individual variants in the same proportion in which they occur in the pooled para-K-casein derivatives. Mackinlay and Wake also made an electrophoretic analysis of the K-casein variants, both before and after rennin treatment, using the urea-mercaptoethanol-starch gel medium. They showed that the major and minor Para-r-casein bands are formed from the individual K-caseins in the proportion in which they occur in pooled para-K-casein. Hence the minor band

130

H. A. MCKENZIE

is not an artifact produced during the alkylation procedure by reaction of iodoacetate with side-chain residues, other than cysteine. Furthermore, Mackinlay and Wake exa.mined the action of rennin on several of the fractions obtained from the chromatography of SCM-K-casein (vide supra). It will be recalled that the electrophoretic pattern of fraction Fahad the B band as a major band and a minor band moving ahead of it. Treatment of this fraction with rennin resulted mainly in the major para derivative together with a small amount of the minor derivative. However, fraction F4,which is enriched in the faster material, gave rise to both para derivatives, but the usual minor derivative is now the major one. The same result was obtained for the B variant series. Thus the material that moves just ahead of the major band for each variant gives rise to the corresponding minor paraSCM-K-casein derivative. This conclusion waa substantiated in a number of other ways. They showed, from a filter paper electrophoretic examination of the macropeptide material released by the action of rennin on each SCM-Kcasein fraction, that only a single macropeptide is released from each SCM-K-casein component. ’ Beeby and Nitschmann (1963) found that the material soluble in 12 % trichloroacetic acid is released from pooled K-casein at an initial rate slower than that of the material soluble in 2 % trichloroacetic acid. As the reaction proceeds the former material is released more rapidly. Also they found that treating K-casein with urea, or repeated precipitation of the protein at pH 4.7, caused the formation of material soluble in 2 % trichloroacetic acid. They concluded that these treatments released the same macropeptide fraction aa rennin does, when acting at low concentration or for a short time. Low rennin concentrations (0.07 pg/ml) released only part of the soluble material from whole acid casein solutions (20 gm/liter) at pH 7. Heating the reaction mixture appeared to “restore” the K-casein, this “restoration” being less complete as the reaction proceeded. On the basis of such experiments Beeby and Nitschmann concluded that K-casein is a complex of proteins bound together by noncovalent bonds. Beeby (1963) postulated that the K-casein “complex” consists of three units, each of similar molecular size (ca. 16,000 minimum molecular weight), and that one of them contains all the sialic acid. Subsequently Beeby (1965a,b) found that, when the pH value of second cycle casein-fraction S is reduced to 3 in the presence of 0.4 M sodium chloride, a precipitate forms; some 20-80 % of the original sialic acid is found in the supernatant, while all the cysteine appears in the precipitate. The supernatant fraction can be separabed into two fractions by chromatography on DEAEcellulose columns. One fraction contains 4-6 % sialio acid, the other fraction contains ca. 0.4-0.6 % sialic acid. After rennin

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131

treatment of the sialic acid-rich fraction, a glycopeptide, soluble in 12 % trichloroacetic acid and containing most of the sialic acid, is obtained. The material obtained after rennin treatment gives two fast moving, positively charged bands and two fast moving, negatively charged bands on urea-starch gel electrophoresis at pH 6.8. Each of the genetic variants of K-casein gives rise to only one of t,hese negatively charged bands on rennin treatment. These results are interpreted by Beeby in terms of his concept of the K-casein complex. Obviously there is a marked discrepancy between Beeby's concept of K-casein-as a complex bound by noncovalent forces that are disrupted by urea, acid, and rennin and containing only one component that is split by rennin, on the one hand, and the views of Wake and his group outlined above. Further work will be necessary, involving careful electrophoretic, ultracentrifugal, and amino acid analyses, if the conflict between these views is to be resolved.

4.

Summary of K-Casein

There has been much progress in the chemistry of K-casein since its discovery by Waugh and von Hippel nearly ten years ago. Nevertheless many aspects of its chemistry remain puzzling and obscure. The reviewer believes it possible to draw certain conclusions from our present knowledge of it : (i) Proteins are by nature sticky substances. They tend to associate to form particles of varying size, ranging from small polymers to large aggregates that may lead to micelle formation, precipitation, or gelation. No protein fraction exemplifies this behavior better than K-casein. The polydispersity, with respect to size, exhibited by solutions of K-casein preparations appears to arise in two ways: first, intermolecular disulfide bonding, which may or may not be present in K-casein as it occurs in milk, but which may arise mainly as a result of oxidation of SH groups during the preparative procedures prior to the isolation of the whole casein fraction from which it is derived; second, a form of noncovalent bonding, which is disrupted readily in solvents containing concentrated urea or formamide, etc. The nature of the latter bonding is puzzling. One might expect at first sight, from the general behavior of casein components as disorderedchain polymers, that it is hydrophobic bonding. However, its temperature dependence does not appear to be what one would expect for hydrophobic bonding. The recent work of Noble and Waugh indicates that, when cu.-casein and K-casein are mixed at 5 O and 20")the larger K-casein polymers are not disrupted and no complex formation appears to take place. If they are mixed at 37", reaction takes place to give what appears t o be an

132

H. A. MCKENZIE

cu,-K-complex of a much smaller size than the K-casein polymers originally present. Similar interaction can be achieved at lower temperatures only if the noncovalently bonded polymers are disrupted first with a reagent such as urea. (ii) All K-casein preparations at present available appear to exhibit chemical heterogeneity as well as heterogeneity with respect to size. The chemical heterogeneity is of two types : contaminating proteins, both casein and noncasein; and heterogeneity that appears to arise when the protein is synthesized in the udder. (iii) It appears, mainly from the work of Mackinlay and Wake, that the latter heterogeneity in K-casein prepared from pooled milk arises as follows. Two types of polypeptide chain (molecular weight 20,000) are synthesized in the udder under direct genetic control. They differ presumably in at least one amino acid residue and this difference appears to arise in the macropeptide portion of the molecule. Varying amounts of carbohydrate are added subsequently to this macropeptide portion of the molecule, Mackinlay and Wake believe that about 10 % of the fundamental chains is altered in the portion of the molecule giving rise to the minor para-K-casein observed during the action of rennin. (iv) Some cows appear to produce only one or the other of these fundamental polypeptide chains of K-casein; others produce both types. It seems highly likely that the genes controlling production of these chains are autosomal alleles, but as yet there is no real genetic evidence of this as insufficient numbers of cows have been examined. ( v ) All fractions of SCM-K-casein appear to be equally effective in micelle stabilization and to be attacked at the same rate by rennin. In the case of SCM derivatives of K-casein, the carbohydrate portion of the molecule does not appear to play an effective role in micelle stabilization or in the enzymatic reaction. However, Thompson and Pepper (1962) have shown that the removal of sialic acid from K-casein by neuraminidase decreases it.s ability to stabilize micelles (cf. Gibbons and Cheeseman, 1962). (ri) The macropeptide, released by the action of rennin, exhibits heterogeneity with respect to solubility in trichloroacetic acid. This appears to be due to the varying amounts of carbohydrate in the peptides released (see also iiz).

H . @-Caseins The procedures developed for the isolation of the clamical @-casein fractions have been discussed generally (Section V,C). Aschaffenburg (1961, 1963b) has shown, by filter paper electrophoretic studies of casein of individual cows in urea solution at pH 7.15, that @-casein

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exhibits genetic polymorphism. Milk from five major British breeds was examined. Three variants, @-caseins A, B and C differing in electrophoretic mobility, were observed. They were found to occur singly or in pairs, as would be expected if each were determined by a single allele. The A variant was the most common and was found in all breeds. Ayrshires and Shorthorns produced no other variant. The B variant occurred in Jerseys, and at low frequency in Guernseys and Friesians. Production of the C variant was restricted to the Guernsey breed, in which it occurred a t low frequency. The significance of these findings in relation to the Channel Island breeds will be discussed (Section V1,C). Aschaffenburg (1963~)developed a procedure for the isolation of the individual variants from milk of cows homozygous for ,%casein. The whole acid casein is dispersed in 3.3 M urea a t pH 7.5, and the pH value is adjusted to 4.6. Under these conditions the bulk of the @-caseinremains in solution while most of the remaining casein fractions are precipitated. Thompson et al. (1962) and Thompson et al. (1964) have confirmed Aschaffenburg’s observations on genetic polymorphism in 8-casehs, using urea-starch gel and urea-acrylamide gel electrophoretic analysis. They examined the casein from each of 1349 cows of five major dairy breeds in the United States. They found that the A and B alleles of @-caseinoccur in Jerseys and Holsteins. The A, B, and C alleles occur in the Guernsey and Brown Swiss breeds. Only the A allele has been found in the Ayrshire breed. Aschaffenburg, Thompson, and their co-workers have made important contributions to our knowledge of the genetic variants of as,l-and p-caseins. However, their recent observation of the nonindependent occurrence of &#,I- and @-caseinvariants is perhaps their most exciting contribution (King et al., 1965; see also Grosclaude et al., 1964). This work has important implications in the synthesis of protein variants in general, as well as of milk proteins. Aschaffenburg and Sen (1963) made a comparison of the caseins from buffalo and cow milk. They found no evidence of genetic polymorphism in the @-caseinof buffalo milk. Thompson and Pepper (1964a) developed a method for obtaining the @-caseinvariants in high purity, as judged by the criterion of urea-starch gel and urea-acrylamide gel electrophoresis. This method consists of fractionation on DEAE-cellulose columns in the presence of 3.3 M urea as dissociating agent. By manipulation of the salt elution gradient it was possible to separate heterozygous mixtures of the @-caseins. Results for the amino acid composition and nitrogen and phosphorus contents of the three genetic variants of @-caseinare shown in Table V-5. The French and Dutch groups used the same methods of preparation

134

H. A. MCKENZIE

as Garnier, Ribadeau-Dumas, and Mocquot (1964a). The nitrogen and phosphorus contents marked with an asterisk refer to these preparations. The nitrogen contents appear to be rather low. These analyses may be contrasted with those of Thompson and Pepper (1964b) for the A, B and C variants: 15.18, 15.33, 15.45 % N and 0,59,0.57,0.50 % P, respectively. TABLE V-5 Amino Acid composition of Bovine p-casehs: Comparison of various workers’ Results (Residues per monomer M.W. 25,000) ~

Variant: Workers.:

@-AB

(GSCM) (PGR)

GlY Ala Ser Thr PTO

Val Ileu

Leu

Phe TYr TV CYS/:!

Met Asp Glu

NHa Arg

His

LY5 Total N (yo) Phosphorus* (%) ~~

@-A

~

5.2 5.6 16.1 10.7 32.8 21.7 10.5 22.1 8.7 4.4 1.o 0 5.7 9.2 39.4 28.6 4.9 5.0 11.1 15.33 0.61

8-B (KR)

5.6 5.4 5.4 5.4 15.4 15.8 9.0 9.5 34.5 34.4 20.6 19.7 10.2 10.0 23.0 22.8 9.3 9.4 4.0 4.0 1.3 1.0 0 0 6.4 6.2 9.4 10.0 40.8 41.5 30.4 4.1 4.1 6.1 6.2 11.7 11.2 14.4* 0.56 ~

(PGR)

8-C

(KR)

5.3 5.5 5.4 6.3 14.4 15.4 9.2 10.1 34.8 33.5 20.4 19.3 10.1 10.2 22.8 22.1 9.4 9.2 4.4 4.6 1.1 1.1 0 0 6.5 6.4 9.3 10.4 40.3 40.0 30.0 4.9 5.2 6.2 6.1 11.6 11.2 14.4* 0.56

(PCR)

(KR)

5.6 5.4‘ 5.3 5.5 16.5 13.9 9.6 9.3 34.2 35.2 20.1 20.0 9.9 10.3 22.9 22.5 9.1 9.3 4.0 4.0 1.0 1.1 0 0 6.0 6.1 9.3 9.7 40.0 41.4 30.6 3.9 4.0 6.4 6.2 13.0 12.2 14.4* 0.56

~

GSCM = Gordon, Semmett, Cable, and Morris (1949); PGR = Pion, Gamier, and Ribadeau-Dumas, de Koning, and van Rooijen (1965); KR = de Koning and van Rooijen (1965). See text for explanation of asterisks. 0

It seems that the genetic variation involves an extra Arg in &casein B and an extra Lys in &casein C as compared with @-caseinA. This has been confirmed by the French and Dutch groups by peptide mapping. Glu is the only amino acid in the B and C variants present in lower amount than in the A variant. Peterson (1965) found that &casein A is hydrolyzed by trypsin at arginine residues only. At low temperature, @-caseinappears to exist in the monomeric form.

MILK PROTEINS

135

As the temperature is raised it polymerizes, the reaction being a slow one in contrast with the rapidly reversible association of a.-casein (see Section V,E). From a study of the sedimentation and diffusion of 8-casein in 6 M urea at pH 7.2 ( I 0.1, POr, NaCl) and in glycinate buffer at pH 12 ( I 0.2), McKenzie and Wake (1959~)concluded that the monomer molecular weight is 19,800 f 1000 and 17,300 f 1000, respectively. It should be stressed that, while concentration dependence of sedimentation and diffusion was allowed for in this work, no allowance was made for selective solvation and the value of ij was assumed to be 0.741 ml/gm. Payens and van Markwijk (1963) have made a study of some aspects of the association of 8-casein. They concluded that the monomer molecular weight is ca. 25,000, that rate of polymerization is slow, and that intertwined thread-like polymers are formed and are interlocked firmly. The extent of polymerization at pH 7.5 and 8.5" was found to be ca. 0.22 and was considerably higher at 13.5'. The value of the second virial coefficient was found by the Archibald sedimentation method to be 7.8 X (gm/dl)-'. This is comparable with values to be expected for rod-like or coiled polymers. Other evidence, especially from optical rotatory dispersion, seems to indicate to the reviewer that the 8-casein exists as coiled polymers. Zittle and Walter (1963) have studied the stabilization of @-caseinto calcium(I1) precipitation at 30" by the presence of K-casein.

I . y-Caseins The classical procedures for fractionation of y-casein have been discussed (Section V,C). More recently Aschaffenburg (1961) has obtained some evidence for the occurrence of genetic variants of y-casein (cf. also the two bands of y-casein observed by McKenzie and Wake, 1959a). Unfortunately, y-casein has received little attention in recent years ; it has seemingly been overshadowed by the search for K-casein fractions. There is some doubt as to whether y-casein is in fact a casein. Very few studies have been made to resolve this issue. One such study was made by Murthy and Whitney (1958), who compared the properties of y-casein with immunoglobulin fractions isolated from mature and colostral milk samples. While the two fractions had somewhat similar movingboundary electrophoretic mobilities, they differed in a number of properties. y-Casein had a phosphorus content of 0.107 yo,whereas none was detectable in the immunoglobulin fractions studied. There were considerable differences in sedimentation behavior. While y-casein is undoubtedly different from the immunoglobulin fractions studied in this work, further studies are needed to make an effective comparison of y-casein with other phosphoglycoproteins of milk.

ASCHAFFENBURG-DREWRY METHOD

1

1

supmaton1 21

precip&lie (casein, fat, etc.)

(whey) (cool to 25", 11 N HCl to pH 2.0)

1

1

I

CL

I

ROBBINSKRONMAN METHOD

I (264 Km/l

1

200

1

I

(262 y/l (NHdzSOd

I

1

1

supernalant

precipitate

(dissolve in equal weight of H,O, add 1 N HCI to pH 4.0 at room temp., hold for 24 hr at 3")

P A

(NE4)ZSOI)

I 1

1

1

supernatant 25

1

precipitate 26

I

precipitate 24

(triturate with pH 5 2 buffer, dialyze

8ime)

a=ainst supernalant 27 (dialyze against HzO)

I

8-laetoglobnlin crystals

precipitate

supanatant

(adjust to pH 6.0 with 1 N NHa, add 500 gm/l (N&)860.)

p-laetalbumin =rum albumin, etc. (triturate with 1/10 whey volume of 0.005 N Nf4, adjust to pH 7.0 with 1NNHa

I

Fractionate for orlsctnlbumin

w

Q,

supernotant (whey)

Precipitate (fat, casein, etc.)

supernatant 23 (Adjust to pH 6.0 with 14 N NK, add

precipitate 22 orlectalbumin, serum albumin, etc. (triturate with 1/10 whey volume of 0.005 N NH8, adjust to pH 7.0 with 14 N NHJ

fractionate for a-laetslbumin

Whole milk

1

supernatant

I

1

precipitole (triturate with pH 5.2

buffer, dialyze)

1

precipitale

I

supaatanf

(dialyze against H20)

1

&lactoglobulincrystals

FIG.VI-1. Outline of the Aschaffenburg-Drew and Robbins-Kronman fractionation procedures. The latter is slightly modified in that the whey fraction is prepared by (NH&S04 fractionation. In both methods the procedure of crystallization has been modified.

X

b

f

(264 gm/l (NH,).SO,)

PreciDitate

-Supernatant 3a a-Lactalbumin, Berum albumin, etc. (triturate with 1/10 whey mlume of 0 . 0 0 5 N N&; adjust to pH 7 . 0 with 1 "H,)

1

Solution Ba Fractionate for a-lactalbumin

(Adjust to pH 6.0 with 1 N mi3)

I

(262 gm/l

A 4

Supernatant 5a

Precioitate 4a

(Triturate with pH 5 . 2 buffer; dialyze

Preciuitate 6a

same' Suuernatant ?a (Dialyze against

i

/3-Ladoglobulin crystals

P r e c i p m 12a

-Supernatant Ila

(DissoIm in 1/10 whey volume of &O; add 0.1 N

tP -3 3*5)

Precioitate 14a

Supernatant 13a

a-lactalbumin, serum albumin, etc. (triturate with 1/10 whey volume of 0 . 0 0 5 N NH,; adjust to pH 7 . 0 withl.O"H,)

(Adjust to pH 6.0 with1"H; add 500 gmri (NH,),SO,)

t

Solution 1Ba Fractionate for a-lactalbumin

4

-Supernatant 15a

sE 2

rn

Precipitate 16a

(Triturate with pH 5 . 2 buffer; dialyze

A

P r e c i p m 180

same)

Supernatant I?a

CI

w

41

138

H. A . MCKENZIE

VI. WHEYPROTEINS A . Ieolation of Major Whey Proteins

It is convenient to consider the isolation of the major whey proteins b-lactoglobulin and a-lactalbumin, together, because most procedures for their fractionation from whey protein have a number of steps in common. In 1934 Palmer (1934) isolated /3-lactoglobulin from bovine skim milk by sodium sulfate fractionation after removal of casein by acid precipitation. (This procedure has been given incorrectly in the review by Tilley, 1960.) Later Sfirensen and Sfirensen (1939) prepared this protein, along with another one called “crystalline insoluble substance,” from an ammonium sulfate whey protein fraction. The latter protein appears to be similar to the protein now called a-lactalbumin. Jacobsen (1949) modified the Sgrensen method so that it was possible for one person to prepare a considerable amount of protein in 5 days. A whey protein fraction, consisting primarily of p-lactoglobulin, a-lactalbumin, the “red” and “green” proteins, and presumably serum albumin was precipitated from an ammonium sulfate whey with ammonium sulfate. The red and green proteins were removed by ammonium sulfate precipitation and p-lactoglobulin crystals obtained by dialysis of the supernatant protein mixture at pH 5.2 against water. This method suffers from the disadvantage that the &lactoglobulin is crystallized from a solution that also contains a-lactalbumin and other whey proteins. The presence of a-lactalbumin tends to interfere with the crystallization of 0-Iactoglobulin and may result in appreciable coprecipitation of impurities. In 1957 a very interesting and valuable discovery was made by Aschaffenburg and Drewry (1957a). They found, by paper electrophoretic analysis, that, if the pH value of a eodium sulfate whey solution is adjusted to 2, a-lactalbumin and serum albumin are precipitated, much of the p-lactoglobulin remaining in solution, They made use of this observation to develop procedures to isolate a-lactalbumin and their then recently discovered bovine p-lactoglobulin genetic variants A and B. Their procedure is outlined (with minor modifications in the final dialyses) in Fig. VI-1. Despite the improvements of the Aschaffenburg-Drewry procedure, it has certain disadvantages. The use of anhydrous sodium sulfate is somewhat inconvenient. It involves the undesirable heating of the milk to 40’. The anhydrous salt dissolves slowly and much stirring is necessary. Careful control of the temperature is needed after cooling from 40’ to avoid crystallization of sodium sulfate in the whey protein solution. The pH value is adjusted early in the fractionation to 2 with 10 N hydrochloric acid, and later to 7 with 14 N ammonia. The danger of local pH excesses during addition of such strong acid and alkali is a real one and must be carefully watched. At the same time, the p-lactoglobulin may be

MILK PROTEINS

139

Whole milk

I

(fat, casein, etc.)

SUP! ;natant (whey) Method Ib: As for Ia, but 11N HC1 and 14 N NH, substituted in titrations Method Ic:

A s for Ia, but pH 2 . 0 precipitation

Method I d

Whey diluted with water 1:l; proceed as for Ib

k

Method Ie:

A s for I&, but pH 3 . 5 precipitation performed at 3’

Method If : As for Ib, but pH 3 . 5 precipitation performed at 3’ Method IIb: A s for IIa, but pH 3 . 5 precipitation performed at 3”

FIG.VI-3. Variation of methods I a and IIa of Armstrong el al. (1966b) for preparation of 8-lactoglobulin and a-lactalbumin.

dissociated at pH 2 to the monomeric form (although this would be partly prevented by the high ionic strength of the solution, see Section V1,C). It seems in terms of current criteria that both the a-lactalbumin and P-lactoglobulin can stand short periods at pH 2 and recover their native state at pH 5-6. However, with increasing sophistication of physicochemical techniques, possibly subtle changes will be detected eventually. Thus it is prcferable to avoid some of the problems of this method. Armstrong and McKenzie (1963) and Armstrong et al. (1966b) studied the fractionation of a-lactalbumin and 8-lactoglobulin, bearing these problems in mind. They started with an ammonium sulfate bovine whey fraction (for the general reasons given in Section 111). Furthermore, the preparation of this fraction can be carried out at 20” without undesirable heating, the ammonium sulfate dissolves readily, and there is no danger of subsequent crystallization in the whey protein solution. They preferred to use 1 N hydrochloric acid and 1 N ammonia for pH adjustment of the pH value, to lessen the danger of local excesses of acid and alkali. Also they endeavored to effect fractionation by lowering the pH value only to 3.5,

140

H . A. MCKENZIE Whey fraction p-lg A milk 100 -@-Lactoglobulin A - a -Lactal bumin -Serum albumin

Method

t

Precipitate

t

Material dial zed S"wrnatant-cmainst DH 2 2 buffer

H IiI

5E5 IIM HCI pH 2.0

A-D

00307 IM HCI pH 35

I0

R

IIM HCI pH 35

Ib

IM HCI pH 35

I U

4

FIG.VI-4. Comparison of Methods Ia, Ib, JIa of Armstrong, McKerieie and Sawyer and the Aschaffenburg-Drewry method. Precipitation is at 20" from the whey protein fraction of milk containing @-lactoglobulinA. The numbem at the top of each pattern indicate the total prot,ein content of fraction per 100 mg whey protein. (After Armstrong el al., 1966b.)

avoiding any danger of dissociation. The fractionations that Armstrong et al. (196613) found most suitable in the preparation of p-lactoglobulin and a-lactalbumin are outlined in Fig. VI-2. Various modifications of these methods, to be discussed below, are outlined in Fig. V I 3 . The distribution of total protein during the fractionation was followed by Kjeldahl nitrogen determinations, and the distribution of kind of protein by starch gel electrophoresis. Typical results are shown in Fig. VI-4. It will be noted that in Method Ia the pH value of the ammonium sulfate whey solution, which has a protein concentration of the same order as that of the original whole milk, is adjusted to 3.5 and a separation of the resulting precipitate made. The precipitate is redissolved to pH 6-7, and the pH value of the supernatant adjusted to 5-6, prior to further fractionation of each fraction. In contrast, the total whey protein is precipitated with ammonium sulfate in Method IIa, and redissolved to 1/10 of the original

MILK PROTEINS

141

whey volume with water. The pH of this solution is then adjusted to 3.5 to divide it into two fractions for further fractionation. Armstrong, McKenzie, and Sawyer showed that the solubility of 0-lactoglobulin is lower at pH 3.5 than at pH 2 in the whey protein- 1.8 M ammonium sulfate solution of Method I (the same being true for sodium sulfate solution). Some of the 8-lactoglobulin is precipitated from the whey mixture along with the a-lactalbuinin and serum albumin at both pH values. The amount of this protein in the pH 3.5 supernatant is less than that in the pH 2 supernatant. However, both supernatants contain virtually only fi-lactoglobulin. Since solutions of P-lactoglobulin alone show no precipitation at pH 3.5 in 1.8 M (NH4)B0,, the precipitation from the whey protein solution must be due to interaction with one or more of the whey proteins or to some influence of the whey medium. The interaction of 8-lactoglobulin and a-lactalbumin has been reported in heated solutions, but no study appears to have been made of interactions under the conditions of the fractionation. The solubility of @-lactoglobulin A is considerably less than that of P-lactoglobulin B at pH 3.5 under the conditions of Method I. Thus the yields of P-lactoglobulin A crystals obtained from Method Ia are low (approximately 0.8 gm/liter). However, there is no significant difference in &lactoglobulin B yield between Method Ia and the AschaffenburgDrewry method (approximately 1.3 gm/liter). Bell and McKenzie (1963, 1964, 1966a) found the solubility of 0-lactoglobulin C to be greater than that of 8-lactoglobulin B, and obtained somewhat higher yields by Method Ia than by the Aschaffenburg-Drewry method. The question arises as to whether Method Ic [(NH4)2S04,pH 21 could be used for preparation of 8-lactoglobulin A to obtain a higher yield without significant dissociation during the period at pH 2, and whether the return to pH 6 is completely reversible. The high ionic strength during the pH 2 period would largely prevent the dissociation. Using present methods for studying conformation and size as criteria of reversibility, the final crystallized material does not appear to be affected by the low pH period. Some workers prefer t o avoid this uncertainty. In Method IIa, very little P-lactoglobulin is precipitated at pH 3.5 with the a-lactalbumin and serum albumin. However, the p-lactoglobulin supernatant contains an appreciable amount of serum albumin and a small amount of a-lactalbumin. While good yields of P-lactoglobulin of high zone electrophoretic “purity” may be obtained, crystallization is more difficult owing to the presence of other proteins. Method IIa can thus be a valuable method for preparation of D-lactoglobulin (especially the A variant), particularly if the remaining impurities are removed chromatographically before crystallization is attempted. Such separations have

142

€I A.. MCKENZIE

been achieved by Armstrong et al. (1966a) on DEAE-Sephadex and Sephadex G-75. The pH 3.5 precipitate from Method IIa is largely free of b-lactoglobulin but contains some serum albumin, so that a solution of this precipitate (19a) (Fig. VI-2) provides a good starting point for the isolation of a-lac talbumin. However, Method IIb (the low temperature version of the pH 3.5 precipitation) (Fig. VI-3) has the added advantage that the precipitate is largely free of both B-lactoglobulin and serum albumin. The solution of this precipitate (19b) is therefore the best starting point for the further purification of a-lactalbumin by salt fractionation or by chromatography. Kronman el al. (1964) have shown that the rate of aggregation of a-lactalbumin at low pH has a positive temperature coefficient, which is consistent with the time dependence of the pH 3.5 precipitation observed in the low temperature procedure (IIb). It is also in accord with the observation of Armstrong, McKenzie, and Sawyer that the pH 5.2 dialyzed solution (fraction 7) in Methods Ic and If (both low temperature methods, Fig. VI-3) contains traces of a-lactalbumin. If Method Ia is being used for the preparation of 8-lactoglobulin and it is also desired to prepare a-lactalbumin, then solution 8a may be used for the latter purpose. Adjustment of the pH of this solution to 3.5 gives a precipitate that may be redissolved to pH 6. The latter solution has virtually the same composition by starch gel electrophoresis as solution 19a in Method Ira, and may be fractionated for n-lactalbumin. The precipitation may also be carried out at low temperature as in IIb, giving a solution on redissolving equivalent to 19b. Armstrong et al. (1966b) consider that adjustment of the pH of ammonium sulfate whey to pH 4 at room temperature does not give a satisfactory separation of the 6-lactoglobulin from the other whey protein. They examined the Robbins-Kronman fractionation and found that the precipitation at room temperature, followed by standing a t 2O, did not result in satisfactory fractionation (Fig. VI-1). In connection with studies of the lactoferrin of milk, Sauchet-Derechin and Johnson (1965a) have shown that it is possible to prepare a-lactalbumin of high electrophoretic purity by chromatography of ammonium sulfate whey on DUE-cellulose. Groves (1965), in studies of the lactoferrin, prepared a-lactalbumin by chromatography of acid whey on DEAEcellulose. In summary, the reviewer recommends the preperation of the whey protein fraction by ammonium sulfate fractionation of milk. It is possible through judicious control of pH, protein concentration and temperature to prepare fractions from which either 8-lactoglobulinand/or a-lactalbumin

MILK PROTEINS

143

of high purity may be readily prepared. I t is furthermore possible, as will be seen later in this section, to prepare not only the bovine proteins but also the proteins of other species, providing careful attention is given to differences between the species. The ammonium sulfate whey fraction may be used for preparation, inter uliu, of lactoferrin, serum transfenins and serum albumin. Most of the methods discussed require some pH adjustment during the fractionation. It would be ideal to avoid these pH changes and to maintain the pH near 5. The reviewer and his colleagues are at present working t o this end.

B. Methods of Zone Electrophorelic Analysis of Whey Proteins During the first twenty years after the discovery of @-lactoglobulinmany investigators studied its apparent heterogeneity, first with phase solubility tests and later with moving-boundary electrophoresis. In 1955 Ogston and Tilley (1955) studied samples from milk of individual cows and found differences in them by these methods. In the same year, Aschaffenburg and Drewry (1955) applied the then comparatively new method of zone electrophoresison filter paper to the study of a sodium sulfate whey protein fraction of milk from individual cows. They found that some samples contained a fast moving 8-lactoglobulin, some a slow moving p-lactoglobulin, and some a mixture of the two. From these observations they were led to propose that the formation of these proteins is controlled by a single gene. Thus this method enabled a very important discovery to be made concerning the heterogeneity of a whey protein. The method was widely used in the study of whey proteins for some years. It is not surprising,. however, that with the remarkable success of the Smithies (1955) method of zone electrophoresis in starch gel, numerous workers applied the method to the study of the whey proteins. Ogston and Tombs (1957), Moustgaard et al. (1960), and Pierce (1961) applied the method to purified 8-lactoglobulins and whey proteins, respectively, but failed to detect further heterogeneity. Bell (1962) reported a study of the development of a starch gel electrophoretic method for whey proteins. The method of starch gel electrophoresis for detection of whey protein t.ypes, as he described it, offers a number of advantages over the filter paper electrophoretic method. It can be applied directly to samples of skim milk without prior separation of the whey proteins. The preparation of the concentrated whey protein solutions required for the filter paper method involves two precipitations and dialysis, with the preparative process taking about 1 day. Furthermore, volumes of 20-50 ml of milk are required and the whole procedure is laborious when many samples are being examined. Bell’s method is simple (providing the compositions of the gel buffer and

144

H. A. MCKENZIE

(a 1 FIQ.VI-5. Starch gel electrophoret,ic patterns of (a) bovine skim milk samples from individual cows. The p-lactoglobulin types, determined by this method, are compared under the patterns with the types indicated by the filter paper electrophoretic method on concentrated whey protein solutions prepared from the same skim milk samples (buffer: NaOH-HsBOs, pH 8.5; voltage gradient: 7.5 V cm-*; time: 5 hours). (After Bell, 1964.) (b): (I) ovine whey proteins Band 3: wrum albumin, Band 2: 8-lactoglobulin, Band 1: 8-lactoglobulin; (119 ovine @-lactoglobulinB. (c) : ovine skim milk samples containing (I) p-lactoglobulin A, (11) @-lactoglobdin A, B, (111) p-lactoglobulin B. For (b) and (c), buffer: Z 0.2 phosphate, p H 7.6;voltage gradient: 7.5 cm-3; time: 5 hours. (After Bell and McKenzie, 1964.)

starch are controlled carefully), requires only a small sample (1-2 ml), and gives good resolution. Evidence of the latter is most strikingly demonstrated by the fact that it enabled him to detect a new &lactoglobulin variant (see Fig. VI-5 and Section V1,C). Bell made a careful study of the kind of hydrolyzed starch used in preparing the gel. He hydrolyaed a number of commercial samples of potato

145

MILK PROTEINS

(b)

(C)

FIG.VI-5 (cont.).

starch and compared the products with the hydrolyzed starch distributed by the Coilnaught Laboratories (Toronto, Ontario). He found that gels prepared from hydrolyzed material from one Australian commercial potato starch gave resolution, for bovine skim milk proteins, consistently superior to the Connaught starch or other preparations. He used a boric acidsodium hydroxide buffer system of pH 8.6 and found that the concentrations of boric acid and sodium hydroxide needed careful control for optimum resolution. It was the failure to realize the importance of this factor that led Bell to report erroneously in his preliminary communication that the A, B and C variants could occur simultaneously in the one milk sample. Bell’s method has several disadvantages, the principal one being that optimum resolution is achieved only with special starch, and that a pH 8.6 buffer system is used. Under these conditions O-lactoglobulin can be denatured and prolonged contact with this buffer can cause complexities with isolated 8-lactoglobulins, as will be seen later in this section.

146

H. A. MCKENZIE

When Bell’s procedure was applied to the milk of other ruminants the resolution of whey proteins was not as satisfactory. Accordingly other buffer systems were developed. Bell and McKenzie (1964, 196613) reported the use of a phosphate buffer of pH 7.6 for obtaining good resolution of ovine whey proteins (see Fig. VI-5 and Section V1,C). Later Bell (1965) developed a starch gel procedure for ovine whey proteins, using the buffer system of Kristjansson (1963). This buffer system gives superior resolution for the ovine proteins. However, its resolution is inferior to the pH 8.5 borate buffer for bovine whey proteins. Peterson (1963) applied the acrylamide gel method to the electrophoretic analysis of p-lactoglobulins and a-lact,albumin, using Bell’s pH 8.6 buffer. He used samples of 8-lactoglobulins A and B provide$ by Townend and a sample of the C variant prepared by Bell. In the course of a study of the alkaline denaturation of 0-lactoglobulins, McKensie and Sawyer (1964, 1966a) observed a pronounced tendency for p-lactoglobulins A, H and C to dissociate near pH 8.5. A similar observation was made independently by Georges and Guinand (1960) for p-lactoglobulin B. McKenzie and Sawyer decided to investigate more closely the splitting of 8-lactoglobulin bands observed occasionally during electrophoresis in starch gel at pH 8.5. They found that, if a solution of the @-lactoglobulinin 0.05 M NaCl (ca. pH 5.2) or a freshly prepared solution of the p-lactoglobulin in the pH 8.5 buffer were applied to the gel, a single diffuse band was obtained for each p-lactoglobulin. The order of spreading of the bands was closely related to their tendency to dissociation at pH 8.5. When solutions of the p-lactoglobulins in the pH 8.5 buffer were allowed to stand for short periods of time, even 0.3 hour, a secondary band appeared in the subsequent electrophoretic pattern moving behind the main band. With increasing periods of time of standing at pH 8.5 prior to electrophoresis, the secondary band increased to a maximum intensity. After 16 hours a series of tertiary bands developed in the subsequent electrophoretic pattern. The magnitude of this effect can be gauged from the patterns for /3-lactoglobulins A, B and C shown in Fig. VI-6. These solutions of pH 8.5 stood for 64 hours prior to electrophoresis. McKenzie and Sawyer had reason to believe, from their other studies of denaturation in alkaline solution, that this effect involved the oxidation of SH groups in the &lactoglobulins. Thus they allowed p-lactoglobulin A to stand in solution at pH 8.5 for 64 hours in the presence of (i) sodium p-chloromercuribenzoate, (zz) phenylmercuric acetate, and (zzi) N-ethylmaleimide. They found that the latter completely blocked the appearance of the secondary and tertiary bands, whereas the former two reagents did not block the reaction completely. Akroyd (1965) has studied the appearance of what appear to be similar bands in acrylamide gel electrophoresis of

147

MILK PROTEINS

(bl

(a)

FIG.VI-6. Starch gel electrophoretic pattern of (a) p-lactoglobulins A, B and C after standing in pH 8.5 buffer for 64 hours, (b) p-lactoglobulin A after standing in buffer for 64 hours in the presence of (I) p-chloromercuribenaoate, (11) phenylmercuric acetate, (111) N-ethylmaleimide (buffer: NaOH-H,B03, p H 8.5; voltage gradient: 8 V cm-l; time: 6 hours). (After McKenzie and Sawyer, 19668.)

0-lactoglobulins, after standing in a pH 8.7 buffer (Tris-HC1). He also suggested involvement of the SH groups. McKenzie and Sawyer stress that, in starch gel elect.rophoretic studies of 8-lactoglobulins, the sample should not be allowed to stand for any length of time in pH 8.5 buffer prior to analysis. In general it is preferable to make up the solution of samples in 0.05 M NaC1. (This problem does not arise, of course, in tlhe analysis of skim milk samples where the skim milk is applied as such.)

C . @-Lactoglobulins 1, Species Diflerences and Genetic Variants

a. Genetic Variants of Bozine &Lactoglobulin. Chemists studying P-lactoglobulin were puzzled for some time after Palmer’s original isolation of it in 1934 by the degree of heterogeneity in a crystalline protein considered to be a “pure” protein par excellence. It was not until 0-lacto-

148

H. A. MCKENZIE

globulin came of age that Aschaff enburg and Drewry (1955) made a brilliant discovery that revealed the major cause of this heterogeneity. It was pointed out (in Section B) that they found 8-lactoglobulin to occur in the form of two variants differing in electrophoretic mobility at pH 8.6. The genes controlling the &lactoglobulin synthesis were shown by Aschaffenburg and Drewry (1957b) to be autosomal alleles without dominance. The variants occurred either singly or together. In their 1955 communication they designated the two variants PI- and 02-lactoglobulin,following a nomenclature suggested by Polis et al. (1950) for fast and slow moving material that they observed in movingboundary electrophoresis of pooled &lactoglobulin. It was found subsequently that there is no simple relation between the and 82 material of Polis et al. and the genetic variants of P-lactoglobulin. It is of interest to note that, while studying different @-lactoglobulins,Klostergaard and Pasternak (1957) identified correctly the genetic variants in t.heir electrophoretic studies. However, in their sedimentation studies, they identified incorrectly the two variants. (The reader is referred for information on the earlier work on o-lactoglobulin to the comprehensive review of Tilley, 1960.) Subsequently Aschaffenburg and Drewry (195713) called the genetic variants p-lactoglobulin A (instead of PI) and @-lactoglobulinB (instead of &) to bring the notation into line with genetic usage. Cows were then described as of phenotype P-lactoglobulin A/A, B/B, or A/B and of genotype Lg*/LgB, etc., using Lg as the locus symbol for the single gene involved. They examined the milk of some 278 cows of Shorthorn, Friesian, Guernsey and Ayrshire breeds and found good agreement between the observed and expected frequencies of the P-lactoglobulin types. This work was confirmed subsequently on herds in Australia (Bell, 1962; McKenzie and Wiley, 1958), in Denmark (Moustgaard et al., 1960), and in t,heUnited States (Plowman et al., 1959). In 1962 Bell showed that certain Jersey cows produced milk that had a genetic variant of &lactoglobulin different from A or B. Bell (1962, 1963a, 1964, 1966) designated this third type as P-lactoglobulin C and assigned the symbol Lgc to the gene responsible for its production. He found that each of the A, B, and C types of bovine &lactoglobulin occurs either singly or together with either of the other two proteins in the milk of individual cows. (Due to an artifact he thought initially that some samples contained all three variants, see Section V1,B.) All six possible &lactoglobulin types were found and no milk sample has been examined in which A, B, and C are absent. When two of the @-lactoglobulinsare present in milk, they occur in approximately equal concentrations. NO case has been observed where the daughter does not contain a 8-lactoglobulin protein in common with the dam.

MILK PROTEINS

149

The foregoing results suggested that the genes controlling the synthesis of @-lactoglobulinsA, B, and C are alleles. This hypothesis was tested in five Jersey herds by the application of the Hardy-Weinberg law, assuming random mating with respect to this character and the absence of strong selective effects. The gene frequencies were obtained by counting, assuming that the observed phenotypes correspond to the genotypes. In all five herds, there was good agreement between the observed numbers and those expected from the theory of genetic equilibrium as indicated by the x2 test. The occurrence of the @-lactoglobulinswas studied by Bell (1964) in the Jersey, Guernsey, Australian Illawarra Shorthorn (A.I.S.), Ayrshire, and Friesian breeds. Using the starch gel electrophoretic method, milk samples from 928 purebred cows were examined. @-LactoglobulinsA and B were found in all five breeds while @-Coccurred only in milk of Jersey cattle. It was considered of importance to determine if the mutation resulting in P-lactoglobulin C is restricted to the milk of Australian Jersey cattle. Using a sample of crystalline p-lactoglobulin C (isolated by Bell and McKenzie) as a reference, Aschaffenburg (1963s) examined the milk from each of 80 British Jersey cows. He found only two samples containing 8-lactoglobulin C variants and these were heterozygous (B-C and A-C). Therefore, 8-lactoglobulin C has not originated in a mutation from Australian Jersey cattle. Since Bell’s present work was carried out, this variant has been detected in a Jersey herd in the United States by Townend and Kiddy following suggestions by Bell (196313). A situation similar to the @-lactoglobulin polymorphism exists with regard to the @-casein of cow milk. Aschaffenburg (1961, 1963b) has demonstrated the three @-caseinvariants A, B, and C in the milk of major British breeds. The polymorphic types are not universal, as only the one variant A was found in Ayrshire and Shorthorn breeds. The Jersey, Guernsey, and Friesian exhibited the second variant B. The third variant C was found only in the milk of the Guernsey. This was the first report of the polymorphic protein difference between the Jersey and the Guernsey. If the &casein C allele in Guernsey milk is of ancient origin, it would point to differences in derivation of the Guernsey and Jersey breeds. The restriction of 8-lactoglobulin C to the Jersey breed is important as it represents a further genetic difference between the Channel Island breeds, which could be used in determining their origin. b. Species Diflerences. Bell and McKenzie (1964) made a comparative study of the whey proteins from milk of a number of species. They found no polymorphism of @-lactoglobulinin goat milk, two polymorphic forms of 8-lactoglobulin in sheep milk, and no @-lactoglobulinin human milk. The boric acidsodium hydroxide buffer of pH 8.5, used in the bovine

150

H. A. MCKENZIE

8-lactoglobulin electrophoretic work, was not satisfactory for use with ovine milk. They were able to demonstrate the presence of two 8-lactoglobulins by using a phosphate buffer of pH 7.6 ( I 0.2). There were protein zones in three regions of the starch gel pattern of ovine whey samples (see Fig. VI-6). The fastest zone (1) was shown to contain serum albumin, the next zone (2) contained a-lactalbumin, and the slowest zone (3) contained @-lactoglobulin. Some samples contained two bands in zone (3). Each of these wm shown to be a /3-lactoglobulin. Genetic studies by Bell (1964) indicated that the genes controlling production of the &lactoglobulins were alleles. The faster moving protein in starch gel at pH 7.6 has been designated ovine B-lactoglobulin A and the slower one ovine 8-lactoglobulin B. Subsequently Bell and Stormont (1965) developed an improved method of starch gel electrophoresisfor examination of ovine whey proteins. They confirmed the earlier genetic studies of Bell. Sen and Chaudhuri (1962) investigated sodium sulfate whey solutions of 23 breeds of Indian goats by filter paper electrophoresis a t pH 4.9 (acetate buffer, I 0.05). They found no evidence of polymorphism for either caprid a-lactalbumin or 8-lactoglobulin, both of which they isolated. Subsequently Bell and McKenzie (1964) reported a starch gel electrophoretic study of skim milk samples of 30 Saanen goats. No polymorphism of 8-lactoglobulin or a-lactalbumin was detected. The reader is referred for further information on these and other genetic variants of milk proteins to the recent reviews of Aschaffenburg (1965) and Kiddy (1964) (see also the important comparative biochemical studies of Sloan et al., 1961). 8. Molecular Size and Confornation

a. Introduction. Pedersen (1936) made an uhracentrifugal study of the “lactoglobulin of Palmer” and found that it had a sedimentation coefficient (3 S) similar to that of the p-peak in dialyzed skim milk. It was this identification that later led Cannan et al. (1942) to propose the name @-lactoglobulin. Pedersen found the sedimentation coefficient to vary between ca. 2.7 and 3.2 S over the pH range 1-10, decreasing from 3.2 to 2.7 S from pH 6 to 8. Pedersen obtained a value of 40,700for the molecular weight in this pH range from s and D measurements. However, from sedimentation equilibrium measurements, he obtained a value for &lwof 37,600 at pH 6.4, but one of 50,200 a t pH 9.8. His measurements showed that a much longer time was required for sedimentation equilibrium to be attained a t pH 9.8 than a t pH 6.4, since a slow aggregation was occurring a t pH 9.8. This aggregation was not noticeable during the short time of

151

MILK PROTEINS

sedimentation velocity measurements. His electrophoretic measurements indicated that a transformation occurred in the molecule near pH 7. Most other workers, in the fifteen years following Pedersen, obtained values for the molecular weight of a similar order of magnitude, although nearer to 36,000. However, one set of measurements stands in marked contrast with the rest, namely, those of Bull (1946) by the surface film method. He studied monolayers of 8-lactoglobulin spread on 20 % ammonium sulfate, obtained an average molecular weight of 17,000 and concluded that under these conditions 0-lactoglobulin was dissociated into two fragments. Bull suspected that heavy metal cations may have a pronounced effect upon the gaseous films of 8-lactoglobulin. He noted, “ I n previous measurements the trough had in it an outlet which was stoppered by a brass screw. In spite of careful coating of the trough with wax, it was noted that this screw had become eroded. At the beginning of the present study this screw TABLE VI-I Injluenee of Cupn’c Sulfate on Molecular Weight and Area of Gaseous Films of @-LactoglobulinSpread on 23% Ammonium Sulfate So~ulionsa Molar concn. of cuso4

0.00

1.25 x 2.50 x 2.50 x 3.75 x 3.75 x 5.02 x 6.27 x 7.52 x a

10-4 10-4 10-4 10-4 10-4 10-4 lo-‘ 10-4

Area (sq. meters per mg)

Mol. wt.

1.28 1.32 1.36 1.45 1.28 1.45 1.37 1.44 1.44

16,300 17,900 30,800 30,400 32,400 35,200 31,600 37,200 32,800

From Bull (1946).

was removed and replaced by a nonmetallic plug so that all experiments reported in the present paper were done under conditions which excluded contamination by heavy metal cations. A series of solutions of ammonium sulfate containing 23 yo of the salt was prepared. Increasing amounts of cupric sulfate were added to these solutions and the p-lactoglobulin solutions spread on these ammonium sulfate solutions and the properties of the gaseous protein films studied as a function of the cupric sulfate concentration.” Bull obtained the results shown in Table VI-1. It is apparent that the dissociation of &lactoglobulin is prevented by an appropriate level of copper(I1) under these conditions. This work of Bull is of considerable importance in that it was the first clear evidence for

152

H. A. MCKENZIB

the dissociation of &lactoglobulin from the 36,000 molecular weight into the 18,000 unit. It appears to have been overlooked by many subsequent workers. The role of copper(I1) is also of significance. In view of this and the known preferred groups for copper(I1) binding to ligands and the tendency for &lactoglobulin to undergo dissociation and conformational change in the pH region where imidasole groups and the masked carboxyl group (see below) are being titrated, the reviewer is of the opinion that imidazole groups and/or the masked carboxyl groups may be involved in the region of contact Eetween the two p-lactoglobulin monomers. A decade after Bull’s work, Townend and Timasheff (1957) made the important observation that 8-lactoglobulindissociated at low concentration into monomers of molecular weight 18,000 a t pH values below 3.5 A host of data, accumulated by various workers in the eight years since their observation, indicates that a given Blactoglobulin genetic variant consists of two identical chains, each of molecular weight 18,000. Furthermore we shall see, later in this section, that &lactoglobulin is capable of dissociation over the whole pH range of its existence. In order to understand the conformational and association-dissociation properties of B-lactoglobulins, it is important to know something of their titration behavior. Outstanding work on the titration properties of /?-lactoglobulinhas been carried out by Tanford and his school. b. Titration Behavior. The interpretation of hydrogen ion titratio n curves of proteins in general and of &lactoglobulin in particular was reviewed in this series by Tanford (1962). Complete titration curves for @-lactoglobulin were first determined by Cannan et aE. (1942). Later Nozaki et al. (1959) redetermined the curve for mixed 8-lactoglobulin, and Tanford and Nozaki (1959) determined curves for the A and B bovine variants. Work is currently in progress in the reviewer’s laboratory on variants from a variety of species. Tanford and his colleagues showed that the isoionic points of @-lactoglobulins A and B in pure water were 5.35 and 5.45, respectively, and that they decreased when potassium chloride or calcium chloride was added owing to the binding of potassium(1) or calcium(I1). The titration curves were reversible between the acid end point and pH 9.7. A configurational change was found to occur a t pH 7.5. Titration curves extrapolated to eero time were obtained above pH 9.7. The zero time curves are in agreement with those expected for rigid compact molecules. The curves obtained at infinite time (denatured protein) are typical of flexible polyelectrolyte molecules (see also Tanford and Swanson, 1957). Tanford found that the difference between the curves (native and denatured) lies primarily in steepness rather than in the count of groups. The groupcounting results are summarized in Table VI-2. The most significant

153

MILK PROTEINS

feature of the analysis is that the native protein appears to contain six imidamle groups per two-chain molecule of weight 35,500, compared with the analytical figure of four. The number of carboxyl groups titrated is less than the analytical figure by two groups. After denaturation the group count agrees with the amino acid analysis. It is evident that two of the carboxyl groups of the native protein are titrated with a pK characteristic of imidamle groups. (See also infrared evidence of Susi et aZ., 1959.) It will be noted that this effect is common to both A and B variants and thus the extra two carboxyls of A (see Section VI,C,5) are not involved. TABLEVI-2 Group Counting for Bovine B-Lacbslobulin" Amino acid andysid' Type of group

(u-COOH

Side-chain COOH Imidaeo1e a-N Hz

Thiol

Phenolic Side-chain NHz Guanidy1

Z.V+

A

B

2 52

50

4

4

2 2

2

8 28 6

8 28 6 40

40

'1

2

Titration curve= Native A

Native B

Native AB

Denatured

52

50

51

53

6

6 2

6 2

40

(34)

2

-

40

-

-1

(0)

-

40

AB

4

2 (2)

{(g 40

After Tanford (1962).

* Gordon el al. (1961), Piez et al. (1961).

The figures have been adjusted to the nearest even integer for a two-chain molecule of molecular weight 35,500. c Titration data of Nozaki et al. (1959), Tanford st al. (1959), and Tanford and Nozaki (1959). d Figures in parentheses are subject to considerable uncertainty.

c. Molecular Size in the p H Range 1.85.6. Townend and Timasheff (1957) first reported on the dissociation of mixed bovine j3-lactoglobulin below pH 3.5. Subsequently Townend et aZ. (1960a) measured the equilibrium constant for the dissociation by light scattering, and Timasheff and Townend (1961b) studied the dissociation of the A and B variants. Timasheff and his collaborators found that the sedimentation coefficients of pooled &lactoglobulin, of fl-lactoglobulin A, and of p-lactoglobulin €3 decreased with decreasing pH as the pH value was lowered from 6 to 1.8, as shown in Fig. VI-7. They also found that the intrinsic viscosity remained constant over this pH range. Thus it seemed that dissociation increased with decreasing pH. Measurements of weight average molecular weight by the Archibald sedimentation method and by the light scattering method

154

H . A. MCKENZIE

2.8

-3

I

2.6 2.4

A

/

-

A:

-

I

p

/'Pooled

oi

A

2.2 -

2.0

2

3

5

4

6

PH

FIG.VI-7. Plot of Sedimentation coefficient (sro,,,.) vs. pH value for bovine 8-lactoglobulins A and B, concentration 10 gm/l and Z 0.1 (NaC1-HCI) : A, @-A,2"; A,8-B, 2"; 0 , p-A, 25"; 0 , p-B, 25'; dashed line, best line drawn through points at 25" for pooled 8-lactoglobulin A, B. (After Timasheff and Townend, 1961b.)

support this hypothesis. The single peak observed in sedimentation velocity measurements and the effect of concentration on the sedimentation coefficient indicate that the equilibrium is of the rapid monomer-dimer type (see Section IV). The light scattering data are related to the weight average molecular weight, Mw, by 1 1 -Bw/M HC = [l 2BoC] AT

1M

+

+

U

W

where H is an optical constant, M is the monomer molecular weight, C is the total protein concentration (in gmlliter), 2Bo/M is the second virial coefficient of the monomer and AT is the excess turbidity of the solution over the solvent. The equilibrium dissociation constant, K d (moles liter1), is related to Bw by 2C(2M - J z w ) 2 Kd = (2)

M'B,

- M'

Typical values obtained by Timasheff et al. for K d and various thermodynamic functions are shown in Table VI3. It is stated frequently by workers in this field that @-lactoglobulindis-

155

MILK PROTEINS

sociates below pH 3.5. This is a half truth capable of causing bitter disillusionment. It is important to realize that the degree of dissociation, a! (weight fraction of protein dissociated), is strongly dependent on the taken from Timasheff and Townend concentration. Plots of a and Sw, (1962), are shown in Fig. VI-8. Since the general sedimentation behavior of 8-lactoglobulins A and B at low pH is indicative of a rapidly associating-dissociatingsystem of the monomer dimer type, Gilbert and Gilbert (1961; see also Gilbert, 1960) made a quantitative comparison of theoretical s vs. C curves with the experimental data of Timasheff and his collaborators. The curves were TABLE VI-3 Dissociation of &Ladoglobulins A and B at pH 8.7"

Protein A

B

K d

AF"

Temp. ("C)

(mole/l

(kcal/ mole)

4.5 8 15 25 4.5

2.84 f 0 . 6 4.08 f 1 . 0 7.50 f 0 . 6 13.0 f 2 1.00 f 0 . 5 1.44 f 0 . 6 2.43 f 0 . 9 5.08 f 1 . 2

8

15 25

x

106)

5.8 f 0 . 2

t.7 f 0.3 5.5 f 0.1 5.3 f 0.1 6.4 6.2 6.1 5.9

AH"

(kcal/ mole)

AS" (e.u.)

12.4 f 1 . 4 23.8 f 5

f 0 . 3 12.8 f 0 . 9 23.2 f 3 f0.3 f0.2 f0.1 -

2RolMilr (l/w X 10-7) 4.6

4.8 7.0 6.9

Data of Timasheff and Townend (1961b).

calculated on the assumption that (i) so for the monomer is 1.89 S, (ii) the dimer moves faster than the monomer by the factor PI3 because of its weight and 1/1.044 because of its shape (i.e., s2 = 1.52 sl), (iii) s for each species considered alone obeys the relation, s = SO [1/(1 gC)] where g is a constant and (iu) g is 0.1 (gm/dl)-l. The curves are shown in Fig. VI-9. It is seen that there is good agreement a t pH 1.6, but an obvious unexplained discrepancy a t pH 3.5. It is important to realize that the shape of these curves is due to the opposition of two factors: (i) the increase in relative sedimentation velocity of the protein as a whole, as the average particle size increases with concentration, and (ii) the normal decrease in velocity caused by changes in the viscosity and density of the solution and back-flow of solvent. Recent work in Canberra indicates that other variants of p-lactoglobulin dissociate a t low pH under conditions similar to those for bovine B-lactoglobulins A and B. Green and Aschaffenburg (1959) have determined the geometric structures of bovine @-lactoglobulinsA and B in the crystalline state. Both

+

156

H. A. MCKENZIE

2

4 6 8 Protein concentration, gm/litar (a)

10

a

02 -

2

4

6

8

10

Protein concentration, gm/liter (b)

FIG.VI-8. Plots of (a) apparent weight average molecular weight (Mw), (b) degree of dissociation (a)vs. protein concentration at pH 2.7 ( I 0.1, NaC1-HCl) for &lactoglobulins A, B, AB(N), and A B calculated from A and B. (From Timasheff and Townend, 1962.)

+

proteins can be described by a structure of molecular weight 35,000 consisting of two similar spheres of radius 17.9 A in contact, with a twofold axis of symmetry between them. Timasheff (1964) has considered possible reasons why these units dissociate a t low pH. Taking charges from the titration curve of @-lactoglobulin(see subsection b above), he calculated the electrostatic free energy of repulsion (AF,r) from the Verwey-Overbeek equation,

157

MILK PROTEINS

where IL0 is the surface potential of the molecules calculated from DebyeHuckel theory, D is the dielectric constant of the medium, b is the radius of the protein unit, R is the distance between the centers of the two spheres, k is the Debye-Huckel screening parameter and y is a function that has been tabulated by Verwey and Overbeek. The results are shown in Table VIA. The sum of AF,l and AFo (the standard free energy of dissociation)

"I

0.5

1.0

1.5

20

Concentration (gmI100 ml)

FIG.VI-9. Plot of sedimentation coefficient (s~g,,,.) vs. concentrationfor bovine 8-lactoglobulin AB at low pH: (a) K d = 2.5 X lo-* mole Iiter-l, (b) Ka = 4.3 X 10-s mole liter1, (c) Kd = 4.3 x lo+ mole liter-1; -, weight average (theory), - -, median of boundary (theory); experimental values of Townend et aZ. (1960a). (After Gilbert and Gilbert, 1961.)

-

gives the total amount of free energy (AF,) necessary to overcome the attractive force between the two spheres. It can be seen that AF. is constant a t ca. 9.8 kcal/mole, AF, was found to be independent of I and D . Timasheff considers that the combination of the two spheres is due to a hydrophobic effect. The effect of electrolyte on the dissociation (increasing I decreases the dissociation) is in agreement with this hypothesis. On the other hand, it could be interpreted in terms of electrostatic effects. Insufficient evidence is presented on the effect of nonpolar solvents on the

158

H. A. MCKENZIE

TABLEVI-4 Dissociation of j%Lactoglobulin A B at Low p H ( I 0.1)"

0

2BoIM112

A(AFed

PH

AF"

(kcal/mole)

1.6 2.0 2.5 2.7 3.0 3.5

4.9 f 0.1 4.9 f 0.1 5 . 5 f 0.2 5.7 f 0 . 1 5.8 X 0.1 7.3 f 0.2

-14.1 -13.6 -10.8 -9.6 -7.9 -4.8

(I/&

-AF,

12.3 x 7.5 x 5.0 x 4.6 x 4.1 x 3.2 x

19.0 18.5 16.3 15.3 13.7 12.1

10-7 10-7 10-7 10-7

10-7 10-7

Data of Townend el al. (196Oa).

reaction to assess unequivocally whether this is a hydrophobic effect or not. Townend et al. (1961) showed that, if @-lactoglobulinsA and B are dissociated at low pH, mixed, and the pH returned to 5.3, in moving-boundary electrophoresis there is no evidence of an AB complex of intermediate mobility between those of A and B. They considered a number of possible models and possible hybrids, as shown in Fig. VI-10. Model

I

I1

IV

111

-2

Identical with origirial molecules

0

FIG.VI-10. Models of plactoglobulins A and B and possible hybrids considered by Townend el al. (1961).

Using a radioactive tracer technique similar to that developed by Itano and Singer (1958) for studying hemoglobins A and S, they showed that Model IV does not apply to 8-lactoglobulin. Much chemical evidence has since accumulated to indicate that Model I is applicable to @-lactoglobulin. This is also in accord with the Green-Aschaffenburg model. d. Molecular Size of @-Lactoglobulins in the p H Range 3.5-5.4. Introduction. Ogston and Tilley (1955) appear to have been the first to examine B-lactoglobulin samples prepared from milk of individual cows. They

MILK PROTEINS

159

distinguished ‘(normal” and “abnormal” types of P-lactoglobulin by the effect of the concentration of the protein on the electrophoretic and sedimentation velocity patterns. It was concluded that one of the species could associate at pH 4.7 and low temperature to a dimer of the 35,000 molecular weight unit. Similar conclusions were reached from sedimentation experiments on pooled P-lactoglobulin by Townend and Timasheff (1956), who found that the pH dependence of the association was “bellshaped” with a maximum near pH 4.5 and falling off to “zero” at pH 3.5 and 5.2. Considerable light was thrown on these observations by two important discoveries. Aschaffenburg and Drewry (1955, 195713) discovered that pooled P-lactoglobulin was a mixture of two genetic variants, A and B (as has been discussed in Section VI,C,la). The second discovery was the theory of Gilbert (1955) for the behavior of associating-dissociatingsystems (discussed in Section IV). Ogston and Tombs (1957) and Tombs (1957a,b) examined the heterogeneity of samples of the A and B proteins and their behavior in transport experiments in the light of Gilbert’s theory. They concluded that their samples of both variants contained about 10 % of a minor component, and that they polymerize at pH 4.6 and low temperature to form a t least a trimer of the 35,000 molecular weight particle. Also they considered that Ogston and Tilley’s “normal” samples of P-lactoglobulin were P-lactoglobulin A, and their “abnormal” samples mixtures of the two variants. Their conclusion that both variants can polymerize at pH 4.6 and low temperature was based primarily on moving-boundary electrophoretic measurements. This conclusion is in error. McKenzie and Smith (1958), Timasheff and Townend (1958), and Townend et al. (1960b) showed that only 0-lactoglobulin A gives evidence of appreciable polymerization in sedimentation velocity patterns under these conditions. The nonenantiographic and bimodal descending patterns that Ogston and Tombs obtained in electrophoretic experiments were also obtained by Timasheff and Townend (1960, 1961a). It is important to note that Tombs obtained similar types of pattern for the B variant at pH values 4.6-5.6. Even the A variant does not polymerize at the higher pH values. Thus the electrophoretic patterns must arise from another cause, e.g., an isomerization reaction. Further studies of the electrophoresis of the B variant are warranted. These should be carried out with a variety of buffer ions and over a range of pH and protein concentration. There is no evidence for the polymerization of the C variant of p-lactoglobulin at pH 4.6 and low temperature from sedimentation velocity measurements (Bell arid McKenzie, 1964, 1966a). McKenzie et at. (1966) obtained some indication of weak polymerization of the B variant from the

160

H. A. MCKENZIE

shape of the szo.w vs. C curve a t pH 4.65. However, Kumosinski and Timasheff (1965) claim that it is possible to detect polymerization of bovine p-lactoglobulin B a t pH 4.6 by light scattering and that "tetramers" of the 36,000 molecular weight unit are formed. The association constant at 4.5". Thus this association is considerably is 6.8 X l o 7 liters weaker than that of the bovine A variant. We shall now consider the association of the latter variant in some detail. Sedimentation measurements. McKenzie and Smith (1958) made sedimentation velocity measurements on bovine 8-lactoglobulins A and B over the temperature range 3-30", at a concentration of ca. 20 .gm/liter in Ogston-Tilley acetate buffer (0.1 2cI NaOAc-0.088 M HOAc) of pH 4.65 (20"). It can be seen from Fig. VI-11 that over the whole temperature range the B variant exhibits a single peak with an szo,wof ca. 2.8 S. In contrast, the pattern given by the A variant depends on the temperature, as was found by Townend el al. (1960b) for pooled 8-lactoglobulin, and Ogston and Tilley (1955) for their "normal" samples. The pattern at 3" is of the typicaI bimodal type predicted by Gilbert for rapidly poIymerizing systems where n > 2 (see Section IV). The szo,w value for the fast peak is 5.9 S. There is less of the fast moving material a t 8.7", the szo,n of the fast peak being 5.4 S. (It is important to note that, although the s vaIues have been presented as s20,w values for convenience of comparison, this must not be taken to imply that, if the experiment was carried out at a temperature other than the original temperature, the S Z O , ~value would be the same in an associating system such as this.) At 20" a single broad peak appears of szo,wca. 3 S and at 29" a single symmetrical peak of szo,v ca. 2.9 8. Since this work was carried out the new bovine genetic variant C has been discovered. Bell and McKenzie (1964, 1966a) showed that it has rather similar sedimentation behavior a t pH 4.65 to that of the B variant. This behavior was confirmed by Townend el al. (1964). Bell and McKenzie (1964, 1966c) showed that the ovine A and B variants behave similarly to the bovine B and C variants. Timasheff and Townend (1961a) have determined the concentration dependence of S ~ Ofor , ~the fast peak of bovine la lac to globulin A at pH 4.65 and 2". Their data were presented earlier in this review (in Fig. IV-4). (It is important to realize, as pointed out in Section IV, that neither the fast nor the slow peaks represent pure polymer or pure monomer.) Gilbert (1963) has modified his earlier theory to include the effect of concentration dependence of sedimentation, but not the effect of diffusion. He has applied his theory to the data of Timasheff and Townend. (A comparison of the theoretical and experimental results is shown also in Fig. IV-4.) In considering these theoretical results, it is important to realize that the following assumptions have been made :

MILK PROTEINS

161

FIG.VI-11. Sedimentation patterns for bovine j3-lactoglobulins A and B a t pH 4.65

= 0.1, CH3COOH-CH8COONa) a t various temperatures (as shown below each diagram). The rotor speed was 59,780 rpm and the phase plate angle was 75”. A,3”: protein conc. = 19.5 gm/l; 108 min (after reaching full speed); ~ 2 0 fast , ~ = 5.9 S, SZ~.,,. slow = 3.6 S. A,Q”:protein conc. = 19.5 gm/l; 90 min; S Z ~ fast , ~ = 5.1 S, szo.w slow = 3.1 S. A,20°: protein conc. = 19.5 gm/l; 107 min; S ~ O = , 3.7 ~ S. A,29“: protein conc. = 19.5 gm/l; 79 min; sro,w= 3.0 S. B,2”: protein conc. = 18.2 g m / l ; 124 min; 8 2 0 , ~= 3.0 S. B,7”:protein conc. = 18.2 gm/l; 124 min; SZO,,,. = 2.9 S. B gives similar patterns at temperatures of 7“ and 30”. (After McKenzie et d.,1966.)

(I

(1) The protein dimer of molecular weight 36,000 is considered not to dissociate to any extent into the monomer of 18,000 under the conditions of the experiment. Thus in the polymerization reaction, dimer is considered to be a‘Lmonomer”(ie., n = 1). (2) Essentially onIy “monomer” (36,000) and “tetramer” (4 X 36,000) are considered to be present. (3) sozo,, for “monomer” is considered to be 2.87 S. (4) The frictional ratio of the 36,000 unit is taken as 1.044. (5) .s’~o., for the “tetramer” is taken as 2.87(42/3)1.044r4,where r4 is the frictional ratio of the “tetramer.”

162

H. A. MCKENZIE

(6) The overall stability constant for the reaction is taken as 5 X 1011 liter3mole-a. What is the justification for these assumptions? (1) It has generally been considered that the 36,000 dimer dissociates only at pH values below pH 3.5. This is erroneous. Georges and Guinand (1960) showed that the bovine B variant can dissociate at pH values above pH 6.5; McKenzie and Sawyer (1966b) showed that all three bovine variants can dissociate over the whole pH range 2-9. The extent of this dissociation depends on the concentration and pH, and the order of dissociation of the three variants is padependent. At pH 4.65 the extent of dissociation is small, and would not greatly afl’ect the determination of weight average properties in light scattering or sedimentation equilibrium experiments a t the concentrations employed in studying the “tetramerization” reaction. However, in transport experiments (e.g., sedimentation velocity and descending limb in electrophoresis, see Section IV), the dissociation could be expected to have a significant effect on the form of the trailing edge. (2) Bjerrum (1941) in his epoch-making thesis showed how the formation of complexes between ions and simpIe ligands is usually stepwise: M+L*MLI MLI

+L

MLz

+ L F? MLa

ML,,-1+ L

MLz

& MLn

The overall stability constant, K,, is given by

In general the formation of complexes does proceed stepwise. However, if steric and bonding considerat,ionsdictate, one or more of the intermediate complexes may be very weak, and the nth complex may predominate. A typical example of this is the iron(I1) complexes of 1,lO-phenanthroline, where the red Tris complex predominates [for reasons discussed by Irving and Williams (1953), Bas010 and Dwyer (1954), and Dwyer and Broomhead (196l)l. Similar considerations on stepwise formation of complexes should apply to proteins. In an elegant piece of work, Witz et al,. (1964) showed by small angle X-ray scattering that the (‘tetramer” of &lactoglobulin is best described by a cubic array of eight spheres (each of these being the

MILK PROTEINS

163

18,000 monomer unit of Green and Aschaffenburg, 1959). Subsequently Timasheft’and Townend (1964) and Green (unpublished work) reached the conclusion that the structure is best described by the cyclic structure shown in Fig. VI-12. While the occurrence of this structure is a strong reason for the assumption that the tetramer is stabilized considerably with respect to the intermediate polymer, it is not a compelling one.

C

D

FIG.VI-12. Staggered structures for the octamer of bovine @-lactoglobulinA: (A) top view, 422 symmetry, d = dyad axis of symmetry, t = octamer bond; (B) side view, 422 symmetry, T = tetrad axis of symmetry; (C) dimer structure; (D) 222 symmetry, X = overall dyad axis of symmetry. The preferred structure is 422. (D. W. Green, unpublished; Timasheff and Townend, 1964.)

Timasheff and Townend (1960) appear to argue in their paper (pp. 3172-3) that the bimodal nature of the sedimentation pattern precludes the existence of intermediate polymers and of monomer and tetramer in equilibrium with hexamer, etc. This is not necessarily the case. The Gilbert (1959) theory does not demand that, when three or more polymeric species are present, the pattern be bimodal. In fact he has shown by numerical examples that the pattern may be bimodal or may be an asymmetrical single peak. (3) The reviewer would tend to place szo.n nearer to 2.95 S, but this is a minor matter. (4) This is in accord with the Green-Aschaffenburg (1959) model. (5) This assumption is in agreement with current models for &lactoglobulin tetramer.”

164

H. A.

MCKENZIE

(6) This value is taken from the light scattering measurements of Townend and Timasheff (1960) and is in agreement with the Archibald and optical rotatory dispersion measurements of McKenzie et a,?. (1966). In their treatment of their sedimentation velocity and light scattering data, Timasheff and his collaborators concluded that the A variant contains 10 % of material that is not able to associate. This conclusion was based largely on Tombs’ sohbility studies, on the considered failure of the area distribution of the fast peak to approach 100 %, and on the agreement obtained for the s vs. C curve making this wumption. Tombs’ data do not necessarily demonstrate that his samples or any other samples of p-lactoglobulin A contain as much as 10 % material incapable of association. Nor were the concentrations of protein in the transport experiments necessarily high enough to approach the asymptotic limit. It is of interest that Gilbert’s recent calculations show that even 10 % of nonassociating material has little effect on the s vs. C curve (as shown in Fig. IV-4). Weight average properties. Another approach to the study of this polymerization is the measurement of weight average properties (see Section IV). Townend and Timasheff (1960) have measured the weight average molecular weights (aw) and the stability (association) constant (K,) for the reaction, making similar assumptions to those discussed above, Their values for the stability constant and thermodynamic functions are shown in Table VI-5. A schematic representation of the pH dependence of the thermodynamic functions, due to Timasheff (1964), is shown in Fig. VI-13. McKenzie et al. (1966; McKenzie and Smith, 1960) made aW measurements for the A and B variants a t pH 4.65 by the Ehrenberg (1957) modification of the Archibald method, using the theoretical treatment of Kegeles and Narasinga Rao (1958). They calculated K,from these measurements, using a modification of the method of Narasinga Rao and Kegeles (1958). The assumptions made in the treatment were similar to those discussed above, except that it was assumed that all the A variant was capable of association. Assumption of 10 % “impurity” gave a poorer fit to the experimental data, but it was not possible to distinguish unequivocally between the presence of 5 % “impurity” and no “impurity.” There is good agreement between the K, values obtained and the light scattering values of Townend and Timasheff, as can be seen from Table VI-5. While carrying out a study of the effect of pH and temperature on the conformation and state of association of proteins, McKenzie and Smith (1958) made some interesting observations on the effect of polymerization of proteins on their optical rotatory properties. They found that considerable changes in optical rotation can take place during association reactions. It was decided to carry out such measurements on bovine

165

MILK PROTEINS

TABLE VI-5

Association Constants and Themnod~amicParameters for the Udamerization of Bwine @-Lado@obulinA at pH 4.86" Archibald Log K . from* ArchiTemp. bald ("C) 1.0 2.2 3.0 4.0 4.5 5.0 6.1 7.0 10.0 10.5 11.0 13.0 15.5 16.0 20.0 20.1 25.0 30.0

(am) - 13.3 -

11.1

9.9 9.4 -

12.3 11.8 11.4 10.8 10.2 9.8 9.2 -

a0

12.6 12.0 11.6

-

11.2

10.7

(Mw)

(AH = -40 Light kcal/mole) sc8tteringb -AF" -AS"

-

11.7

-

-

-

11.1

-

-

10.5

-

9.8

10.1 9.8 9.1 -

-

-

9.2 8.5 7.9

a0

(AH = -64

kcal/mole)

-AF" 15.8 15.2

-

14.8 14.4 13.9 13.3

-ASO 178

-

179

-

179

-

-AS"

-

-

-

14.4

138

-

179

-

179

-

12.9 12.2

178 178

-

-AFO

179

-

-

Light" scattering (AH = -53 kcal/mole)

-

From McKenaie et al. (1966). b K . liter" (base moles)-* Data of Townend and Timasheff (1960).

8-lactoglobulins A and B. Subsequently these measurements were extended to the bovine C variant and the ovine A and B variants as they were discovered. It was also shown how optical rotation and rotatory dispersion measurements can be applied to the determination of K. in favorable cases. This work will now be considered briefly (see also Bell and McKenaie, 1966a,c; McKenzie et al., 1966). If the numerical value of the specific rotation (i.e., the levorotation, denoted here by/l~r]~/) of bovine p-lactoglobulin B at pH 4.65 is plotted against temperature, the curve shown in Fig. VI-14 is obtained. It will be noted that there is a small increase of /[&8/ with temperature, in qualitative accord with the rule of Kaurmann and Eyring (1941), if 8-lactoglobulin has some rigidity of structure. The same kind of temperature dependence is observed for p-lactoglobulin C. In contrast, p-lactoglobulin A has the temperature dependence shown. The marked deviation from the slope observed for B and C occurs only at lower temperatures, in the region

166

H. A. MCKENZIE

-AS -AH -AF 200- 70-

150loo 50 -

50 -

30 -

4 @A =@A,

4.0

; 4.5O 4.5

5.0

PH

FIQ.VI-13. pH Dependence of thermodynamic parameters for the oetamerisatiort (“tetramerization”) of bovine &lactoglobulin A. (After Timasheff, 1964.)

where the “tetramerization” (octamerization) reaction occurs. That this effect is linked with this reaction can be seen from the effect of concentration. Analysis of the optical rotatory dispersion curves shows that they obey the Moffitt-Yang (1956) equation, that a. changes considerably on association, and that the change for bo is small. McKenzie, Sawyer, and Smith have shown that both the change in [a] and a. may be treated as suitable weight average properties to give values of K,. Typical plots are shown in Fig. VI-15. The values for K , are compared in Table VI-5 with values of K , obtained from light scattering measurements and molecular weight measurements by the method of Archibald. It is seen that the agreement is very satisfactory. Thus the measurement of optical rotatory dispersion can, in favorable cases, provide a very useful means of investigation of associationdissociation reactions. Herskovits et al. (1964) have made an independent study of the effect of polymerization on optical rotatory dispersion properties of p-lactoglobulin A. They did not calculate K , values, but used their K , values from light scattering measurements to calculate a. values, which they found to be in good agreement with experimental values of ao. Few studies have been made of the effect of association-dissociation reactions of proteins on their optical rotatory properties. When McKenzie and Smith made their original observations in 1958, they noted that the first observations appear to have been those of Carpenter (1935) on whole casein. He found that /[aIs6/ decreased from 105” to 99” as the concentration was increased from 1.5 to 15 gm/liter a t pH 6.8. As was seen in Section V, the caseins undergo a temperature-dependent association, and

167

MILK PROTEINS 50 40

35 30

25

35

I

I

I

I

I

-

25

u

20

G

u I

I

I

I

'

I

P-A, pH 2.1

P-c

30 m

I

15

30 25

20 15

31.5 1

B

10

1

1

m

t

1

30

1

40

Temperature ("C)

FIG.VI-14. The temperature dependence of [a1578 for bovine 6-lactoglobulins A, B, and C: (a) @-A (11.8 gm/l) at pH 7.5 (veronal, I 0.1) and @-A (7.5 gm/l) at pH 2.1 (HCl-NaCl, Z 0.1); (b) 8-A (10.8 gm/l), 6-B (10.5 gm/l), 8-C (11.2 gm/l) at pH 4.65 (CH3COONa-CH&OOH, Z 0.1); (c) 8-A at pH 4.65 for the protein concentrations given on the curves (gm/l). (After McKenzie et al., 1966.)

it is believed that they possess little helical character and that the associa-

tion appears to involve hydrophobic bonding. Boedtker and Doty (1956) found that gelatin shows a decrease in levorotation on gelling, presumably as it goes from a random coil to a highly associated collagen-like structure. Schellman (195813) observed a change in during association-dissociation reactions of insulin. It is of interest to consider how the changes in optical rotatory properties on association arise. The optical rotation of a protein can be considered to arise from (a) the sum of the interactions between groups present in the backbone structure, (b) the sum of the interactions of side chains with one another, (c) the sum of the interactions of the side chains with the backbone structure, and (d) the inherent rotation of the side chains. The small

168

v;

H. A. MCKENZIE

570 m p -1OOF

ny

''

436 mp

313 rnp

364 mp

1

I

1

(0)

-120

-160

-200

-

-220-

I

I

I

I

I

I

I

FIG.VI-15. Moffitt-Yang type of plots of optical rotatory dispersion data for bovine p-lactoglobulins at pH 4.65 in the temperature range 1-45". (a) @-A: 0 , 1"; A, 5'; 10"; 0,13"; A, 20'; 0 , 3 0 " ; V,45'. (b) 8-B: 0 , 3"; A, 10"; . 20"; ,0,30"; A, 40". These plots are shown for only some of the temperatures a t which they were determined originally, to avoid confusion in the diagrtim owing to crowding of the individual curves. (After McKensie el al., 1966.)

m,

change in bo and the increase in a. on "tetramerization" of bovine &lactoglobulin would seem to indicate that there is little change in fundamental conformation of the protein, but that the change in a. is due mainly to changes in (h) and (c). It is important to consider possible mechanisms of the "tetramerization" reaction. It will be recalled that the reaction has a negative enthalpy and a large negative entropy, so that the polymerization ia favored by low temperature. The pH dependence of the reaction is "bell-shaped," the reaction occurring over the pH range 3.8-5.2 with a maximum near pH 4.5. This pH dependence immediately calk COOH groups to mind as being involved in the reaction (Timasheff and Townend, 1962). It is significant that bovine &lactoglobulin A undergoes this resction and has one extra aspartic acid residue per 18,000 monomer unit (2 per 36,OOO unit-

MILK PROTEINS

169

the “monomer” for the “ tetramerization”) compared with the B variant, which does not undergo the reaction. Bell and McKenzie (1964) found that no other ruminant 0-lactoglobulin undergoes this reaction strongly and that all of them have one less aspartic acid residue per monomer than the bovine A variant (however, vide infra). Armstrong and McKenzie (1964,1966) blocked carboxyl groups in bovine @-lactoglobulin A with l-cyclol~exyl-3-(2-morpholinyl-(4)ethyl)carbodiimide metho-p-toluene sulfonate (hereafter referred to as CMC). Even 30 % (max.) blockage largely prevented the “ tetramerization” reaction from occurring. At 80% (max.) blockage the reaction was prevented completely. There were only small differences in optical rotatory properties between the CMC-modified protein and the native protein. Typical sedimentation patterns are shown in Fig. VI-16.

FIG.VI-16. Sedimentation patterns of bovine 8-lactoglobulin A at pH 4.65 ( I 0.1, CHaCOOH-CHsCOONa) with and without modification of carboxyl groups (speed: 59,780 rpm) : (a,b) no modification, conc. = 15 gm/l (2.5’) for (a) 64 min after reaching full speed and for (b) 120 min; (c,d) max. 30 % modification, conc. = 15 gm/l (2.3’) for (c) 68 min and for (d) 134 min; (e,f) max. 80 % modification, conc. = 15 gm/l, (2.8”) for (e) 64 min and for (f) 121 min. (After Armstrong and McKenzie, 1964, 1966.)

170

H. A. MCKENZIE

Of great interest is Ohe recent discovery of a new bovine &lactoglobulin occurring in the milk of Droughtmaster beef cattle. This variant, P-lactoglobulinDroughtmastcr has been isolated and examined by Bell et al. (1966a). It does not “tetramerize” a t pH 4.6 and low temperature. Its amino acid composition is identical with that of bovine P-lactoglobulin A, but it has sialic acid and hexosamine groups attached to it. These groups occur in a peptide containing, in& aliu, arginine, serine and glutamic acid residues, and not in the peptide containing the extra aspartic acid residue. The carbohydrate groups prevent the association reaction from occurring either by direct blocking of essential carboxyl groups or by steric interference, These observations, taken along with the finding of Bell el al. (1966b) and Townend (1965) of the high concentration of carboxyl residues in the aspartic/glycine difference peptides of A and B, strongly implicate carboxyl groups in the ( I tetramer” bond formation. The most likely type of bond involving carboxyl groups is the hydrogen bond. Mechanisms of homologous hydrogen bond formation involving COOH groups have been considered by Timasheff (1964) and Sherbon and Regenstein (1965). It seems to the reviewer that the most likely mechanism involves the formation of hydrogen bonds between protonated COOH groups in the aspartic acid-glutamic acid residue-rich region of the protein chain surface, As the pH is decreased from the isoionic point, COO- groups become protonated and interact with one another to form hydrogen bonds. Thus one would expect a sharp increase in tetramerization as the pH value is lowered from 5.2 to 4.6. At first sight it might be thought that the free energy would reach a plateau region. However, electrostatic repulsion between the protein molecules increases as the pH decreases: the charge changes from nearly zero at pH 5.2 to ca. +10/36,000 unit at pH 4.6 and to +22/36,000 unit a t pH 3.5. Townend (1965) states that ca. 30 COOH groups are “buried” during the formation of ‘‘tetramer.’’ This would imply a maximum formation of ca. 4 hydrogen bonds per “monomer” during the formation of the “dimer” and “ trimer,” and 8 bonds during the formation of the “cyclic tetramer.” Timasheff (1964) has suggested another mechanism involving the fluctuation of protons on carboxyl groups in the binding sites as these groups are titrated. This schenie is statistically based and gives a maximum formation near pH 4.6 (i.e., near the pK of the COOH groups). It is not possible at present to decide between various mechanisms. Dissociation in the p H region 3.6-5.6. Most workers assumed previously that 0-lactoglobulins can dissociate only in the pH region below 3.5. McKenzie and Sawyer (1965, 1966b) and Bell and McKenrie (1966a) have shown that the bovine variants dissociate appreciably into the monomer (18,000 unit) a t low concentration in the pH range 3.5-9 a t 3-20”. This

MILK PROTEINS

171

dissociation can be detected readily for all the variants, except the A variant in the temperature and pH region of maximum octamerization (4.5-4.7), where the detection of dissociation into the monomer is not so simple. The behavior of the A variant a t pH 4.6 and 20”, where the tendency to octamerization (“tetramerization”) is only moderate, is interesting. At higher concentrations the sedimentation pattern is bimodal, As the value concentration is lowered a diffuse single peak is observed. The szo,w does not increase with further decrease in protein concentration. This behavior suggests that the protein is partly dissociated into the monomeric form. Even at pH 4.6 and low temperature, the C variant shows a single peak at all concentrations examined (up to 50 gm/liter) but there is evidence of association-dissociation, the S Z O , ~vs. C curve of Bell and McKenzie (1966) having the characteristic shape for a rapidly equilibrating system (see Section IV). The discrepancy between these results and those of Townend et al. (1964), who failed to note this effect, is due to the fact that they carried out their measurements only in the concentration range 9-50 gm/liter. Thus their equation relation S2O.w and C is invalid. McKenzie and Sawyer (1965) showed from sedimentation equilibrium measurements by the method of Yphantis (1964) that all three bovine variants are slightly dissociated a t pH 5.4, IO.1, and 20” and that there is little*differencebetween them in their tendency to dissociate under these conditions. At higher ionic strengths the tendency to dissociate is less. e. Molecular Size of /.-Lacloglobulins in the p H Range 6.4-9.2. Pedersen (1936) first found variation in szo,wfor 8-lactoglobulin as the pH was increased above ca. 6-7. Bull and Currie (1946) found that it was not possible to obtain reproducible results in osmotic pressure measurements, if 8-lactoglobulin had been subjected to pH values above 5.8 even for a short time by addition of alkali. Linderstrgim-Lang and Jacobsen (1940) found that 8-lactoglobulin denatured rapidly a t pH 8.3 and 0”. Groves et al. (1951) also studied the reaction above pH 8 and found reversible and irreversible changes that were pH-dependent. Macheboeuf and Robert (1953) and Calvin (1954) concluded that the latter change involved, a t least in part, oxidation of SH groups and rupture of S-S linkages. Thus it would be expected that values of molecular weight determined above pH 6 might be strongly dependent on pH and be time-dependent. Got (1963) obtained evidence of dissociation of 8-lactoglobulin a t pH 7 from molecular weight measurements by the method of Archibald. Georges and Guinand (1960) studied the intensity of light scattered by solutions of bovine 8-lactoglobulin B as a function of pH. (See also sedimentation studies by Georges et al., 1962.) They concluded that this

172

H. A. MCKENZIE

variant is able to dissociate a t pH values above 5.5, and that the dissocia tion increases with increasing pH, ionic strength, and temperature. Unfortunately these workers expressed their results in terms of I vs. C and I / C vs. pH plots rather than using the more usual HC/T term. They conclude that I / C is independent of concentration in the concentration range 1.5-14 gm/liter for a given pH value in the pH range 6-9. McKenzie and Sawyer (1965) and Bell and McKenzie (1966a,c) found that the levorotation of all three bovine variants and both ovine variants increases immediately on mixing at pH values above ca. 6, but that any subsequent change of rotation with time is pH-dependent. It was found did not change with time, and it was considered worth that, at pH 7.5, [a]s7s carrying out sedimentation velocity and equilibrium experiments at that pH value and 20". A single peak was obtained in the sedimentation pattern and the plot of szoZwvs. C had a similar form to that shown in Fig. VI-9 for ,f3-lactoglobulin a t low pH values. The form of the curve at alkaline pH values could be due to an association-dissociation reaction (as discussed in Section IV), and/or it could arise if the conformational change results in a frictional change that is concentrationdependent. Sedimenb tion equilibrium measurements by the Yphantis (1964) method indicated that dissociation was occurring in the low concentration range, the weight extrapolating to ca. 17,000, i.e., close to the average molecular weight, am, monomer molecular weight unit. The value of iI?lwwas concentrationdependent, contrary to the findings of Georges and Guinand. IC waa found by McKenzie and Sawyer that the order of tendency to dissociation is p-A > 8-B > 0-C. The equilibrium constant (K.) for the associationliter mole-'. dissociation reaction of the B variant at pH 7.5 is 1.3 X As the pH increases above 7.5, [aI578changes slowly with time. Plots of ~ 2 0 vs. , ~ C, from sedimentation velocity measurements made immediately after mixing, have a form similar to those a t pH 7.5. After standing for ~ to slow polymerization several days there is a general increase in S Z O ,due of the 8-lactoglobulin, probably involving oxidation of SH gro,ups, an effect already referred to in connection with zone electrophoretic studies of o-lactoglobulin. f. Conformation Studies of p-Lactoglobulin in Solution. If the reviewer had been asked a decade ago to write on the conformation of B-lactoglobulin, he would have confidently written that it was the paragon of the fully helical protein, and in believing this he would not have been alone (cf. Schellman, 1958a). Today he is much less confident aa to what the structures of the various p-lactoglobulins are. However, he would make a number of points to be considered in an ever changing situation. As discussed in Section IV, the occurrence of association-dissociation reactions and of subtle changes in properties of proteins with small changes in ionic

173

MILK PROTEINS

strength, and temperature makes it likely that many proteins exhibit considerable “motility” in native shructure. This is probably well exemplified by the 8-lactoglobulins. The effect of various conditions on [(Y]s~s,on the fitting to the MoffittYang equation, and on values of ao, bo, and X, are shown in Table 1‘1-6. The very low value of [a1678 a t pH 5.2 and the value of A, for the native protein originally led investigators to assume that the helical content of P-lactoglobulin is high. The value of uo possibly indicates a compact structure, and the value of bo indicates a low helical content if it is assumed that only helical and disordered regions are present. In the pH region 2-5.2 there is only a small effect of pH or concentration on the value of [aI678 and a. and bo (except in the region of “octamerization” of bovine &A a t low temperature). As one increases the pH above 5.2 there is an immediate increase in levorotation. Values of [&78 and

451 =t

40

2

3

4

5

6 PH

7

0

9

.

FIQ.VI-17. The pH dependence of the specific rotation of &lactoglobulinsat 578 mp and 20”: B, 8-lactoglobulin A; A, @-lactoglobulinB ; 0 , @-lactoglobulinC. Protein samples were titrated to the appropriate pH value with dilute acid or alkali. Hollow symbols are those points obtained in buffers. Rotations at pH 9.1 were extrapolated to zero time. All concentrations were in the range 10.0 1.0 gm/l. (McKenzie and Sawyer, 1965.)

*

A/[(Y]~,~/for the bovine A, B, and C variants are shown in Fig. VI-17.

It can be seen that A/[CY]~,~/in the pH region above 5.2 depends markedly on the variant and pH (McKenzie and Sawyer, 1966a). The extent of change in [a1578 a t pH 7.5 decreases with increasing concentration and ionic strength, but increases with increasing temperature and depends on the variants being in the order C > B > A (McKenaie and Sawyer, 1965). At higher temperatures there are marked time-

TABLEVI-6 Some Optical Rotatory Dispersion Properties of Bovine fl-Lacbglobulins A , B , and C

PH 5.2

IP

1.8

9.2

Solvent NaCl (I 0 . 1 )

NaCl-HC1 ( T O . l )

NaOH-€l&%-NaCl (I0.1)

Temp. (OC) 25

25

25

Protein concn. Variant

A B G

5

A B C

7.5

q C

5.2

7Mumt+NaCl ( Z O . 1 )

25

C 0

(f) denotes final value.

(gm/l)

5

la1578

(7

Moffit-Yang plot

a0

("1

- 160

-31 .O -34.8 -28.3 -27.6 -29.7

X

< 330 m p

Dep. linearity X

< 330 m p

Changes c time, final linear to X 310 mp Changes c time, linear

-167 -188

-149 -145 -160

bo (")

-72 -70 -65 -75 -73

-75

-275(f)a

-88(f)

-550(f)

-75

MILK PROTEINS

175

dependent changes near pH 7 in lalo, SH availability, and solubility of @-lactoglobulinAB a t pH 5.0 (see the study of Gough and Jenness, 1962a). As the pH is increased above 7.5, McKenzie and Sawyer found that the rotation changes with time a t 20” and that the change is even faster a t 3”. The rate of change and extent of change a t a given temperature are dependent on the concentration. The order for the change is now @-A> 8-B > 8 4 , the latter being much less affected than the other two. At a given concentration and temperature, the kinetics of the reaction are such that two exponential equations are needed to fit the data. Some of the time-dependent changes are linked with oxidation of the more readily “available” SH groups (see Dunnill and Green, 1965) and, at higher pH, hydrolysis of S-S groups. The time-dependent changes are largely irreversible (McKensie and Sawyer, 1966a,b). The effect of urea on the optical rotation of p-lactoglobulin resembles more closely its effect of urea on the optical rotation of bovine serum albumin than on ovalbumin (see, e.g., Kauzmann and Simpson, 1953; McKenzie et al., 1963; Tanford and De, 1961; Christensen, 1951; Johansen, 1951). Typical values for and a. and bo in 7 M urea, pH 6, and 25” are given in Table VI-6. McKensie (1965) has shown that the difference spectrum in the region 220-300 mp, for each of the three bovine variants at pH 2 vs. the protein a t pH 5.2, is developed immediately and does not change with time. At pH 9.2 the difference peaks a t 287, 290 mp do not change with time, but that a t 230 mp does change with time (cf. Pantaloni, 1963). Tanford et al. (1960; see also Tanford et al., 1962) examined the effect of organic solvents on and a. and bo for bovine &lactoglobulin AB. Typical results for dioxane are shown in Fig. VI-18 and Table VI-6. What are the implications of these observations for the conformation of &lac toglobulins? Tanford et al. (1960) concluded from their results that the relatively small value of the levorotation of bovine p-lactoglobulin AB is due in part to the fact that the peptide groups of the protein are buried in the interior of the molecule in a relatively nonpolar environment, with little or no regular order, and that the native structure in aqueous solution is primarily the result of hydrophobic forces. They proposed that on addition of organic solvents a two-step reaction takes place: disruption of the original structure with transfer of unordered peptide groups from the interior into the solvent, and then formation of a new conformation whose optical rotatory dispersion approaches that characteristic of right-handed a-helical polypeptide chains. They suggested that these conclusions were applicable to most other globular proteins. This should be regarded with great caution (cf. Kendrew, 1962; Blake et al., 1965; and Herskovits, 1965).

176

H. A. MCKINZIE

Urnes and Doty (1961) first suggested an alternative explanation for the optical rotatory dispersion properties of &lactoglobulin AB. They pointed out that, if there are equal amounts of helical and &conformations present, bo will be a small number, yet because the contribution of both conformations to [ ( Y I and ~ ~ ~a. is positive these parameters will decrease on unfolding with little net change in bo, corresponding to the behavior actually obis ) served. On this basis they concluded that the fraction of helix ( j ~

ml dioxane per 100 ml totol volume

FIG. VI-18. Effect of addition of dioxane to aqueous solutions of bovine 8-lactoglobulin AB on the optical rotatory properties of the protein at pH 3 and ionic strength 0.02. (After Tanford et al., 1960.)

0.55, the fraction of &conformation (jp) is 0.33, and the fraction of disordered form ( f ~ is ) 0.12 for the native protein. This calculation is in error, however. In reconsidering the optical rotatory properties of P-lactoglobulin, McKenzie (1965) concluded that this suggestion of Urnes and Doty (1961) offers the most reasonable explanation of the data. A calculation by McKenzie indicated that fH is 0.34, fp is 0.34, and fo is 0.33 for bovine &lactoglobulins A, B, and C a t pH 5.2. His examination of the hypochromicity (cf. Rosenheck and Doty, 1961) of these proteins a t pH 5.2 indicated that fH is 0.3. No assumption was necessary about the 8 content, as its contribution to the hypochromicity would be little different from that of the disordered form a t 190 mp. It is of interest that Linder-

MILK PROTEINS

177

strgm-Lang (1955) found the rate and extent of exchange of deuterium with #?-lactoglobinto be low a t pH 5.46. About 330 hydrogen atoms (per dimer unit) exchange instantaneously a t 0”. Of these, ca. 200 are side-chain H atoms; the residual ca. 100 are backbone ones. About 120 react slowly in the range 0-40”. The remaining 100 react only after denaturation. In a recent communication Timasheff and Townend (1965) considered optical rotatory properties of p-lactoglobulins. They stated that the optical rotatory properties observed by others lead to (‘proposals that &lactoglobulin either has almost equal amounts of right-handed and left-handed helices (Urnes and Doty, 1961; Bell and McKenzie, 1964) or that it contains ordered structures other than a-helical (Tanford et al., 1960; Herskovits et al., 1964), e.g., the &structure.” This statement is quite erroneous. Tanford et al., as discussed above, considered that the proposal of equal amounts of left- and right-handed helices did not explain their data as well as their proposal that p-lactoglobulin is disordered in the native state in aqueous solution. They ruled out specifically the consideration of @-structures. Urnes and Doty, and Bell and McKenzie, did not suggest equal amounts of left- and right-handed helices, but considered that the presence of &structure, along with right-handed helices and disordered structure, gave a reasonable explanation of the rotation data (vdesupra). McKenzie has stressed repeatedly in lectures in the United States and Australia during the last two years that this is the most likely explanation. Herskovits et al. concluded in their paper that there is little or no helical content, but that the a0 value indicates some ordered structure. They did not suggest any &structure. (Herskovits and Mescanti, 1965, concluded that @-lactoglobulincontains a ‘(low” or nearly zero “helical content.”) In the same communication Timasheff and Townend considered Cotton effects they observed near 296, 286, 230, and 195 mk in the native protein and the effect of reagents on them. They concluded that the helical content was low and that there was some ordered structure, possibly @-structure. They found evidence of an amide I (C=O stretching) infrared band near 1630 cm-1, a frequency usually associated with the @-structure in polypeptides. The reviewer does not agree completely with their interpretation of the Cotton effects. Furthermore, some of their experimental observations on the Cotton effects are a t variance with observations made by Bodanszky and McKenzie (1965). McKenzie observed the C=O stretching frequency in @-lactoglobulinin infrared and deuterium exchange studies currently in progress. There is evidence from the infrared spectra of the presence of the B-, helical, and disordered structures. However, he would caution against making too facile an interpretation of the infrared data, a point

178

H. A. MCKENZIE

brought home forcefully to him nearly twenty years ago after his early infrared measurements on proteins, and made abundantly clear also by the work of May (1964). Tanford (1961) has made a valuable contribution to our understanding of ionization-linked changes in protein conformation. Tanford and Taggart (1961; see also Tanford et al., 1959) examined the transition in the conformation of pooled bovine /3-lactoglobulin near pH 7.5, in the light of Tanford’s theory. They concluded that a single titratable group is involved and that the group is a “buried” COOH group in the native configuration. Since there are two anomalous COOH groups per 36,000 molecular weight, they concluded that there must be two locations in the molecule where the transition occurs independently. Tanford and coworkers rejected the idea of dissociation occurring in this reaction. However, the work of McKenzie and Sawyer (1965) indicates that possibly the transition proceeds via the monomer. It is of interest that ovine p-lactoglobulin B undergoes this reaction more readily than the other variants: it has one less tyrosine residue per monomer. Dupont (1965a,b) has studied the effect of temperature on bovine p-lactoglobulins A and B, and concluded that the “thermodenaturation” proceeds via the monomer. She concludes that the anomalous COOH group is not involved but that imidazole groups are, on grounds not clear to the reviewer. 3. Phase Solubility Tests for &Lactoglobulin Homogeneity: the Effect of Polgmerization

In the variable solvent solubility test for protein homogeneity (Falconer and Taylor, 1946), a constant amount of total protein is brought to a series of different concentrations of salt, the volume, pH, and temperature being kept constant. Every protein has a characteristic solubility curve and if two or more proteins are present they exhibit a combined curve with sharp breaks, each part of the curve following an equation of the form: S = Soe-kz (or In S = B - kI), where S is the solubility at ionic strength I , and SO, p, and k are characteristic constants for the protein. Each break in the curve represents the appearance of a new solid phase. The break does not necessarily arise from a new protein in solution. If the amorphous form of a protein precipitate is transformed into a crystalline phase during the test, an extra break will appear in the solubility curve. Ogston and Tombs (1956) have shown that, if a single protein in solution is in equilibrium with different solid phases, according to the concentration of salt, a discontinuity can appear in the curve. Fortunately, a distinction between this case and the one where two protein components are present in solution

179

MILK PROTEINS

is simple in principle. Ogston and Tombs showed that, if the amount of total protein is changed, the position of this discontinuity in the former case is invariant. In the latter case the point of discontinuity shifts. Where an invariant point occurs the two solid phases can be due, for example, to solid protein and solid protein combined with a solvent component. There is another possibility not considered by Ogston and Tombs. If there is a single protein present and it undergoes a polymerization reaction, only one or several solid phases may separate. It follows directly from application of the phase rule that an invariant point may occur providing monomer and polymer are in equilibrium, monomer and polymer have separate solubility curves, and there are two solid phases corresponding to monomer and polymer. In the constant solvent phase solubility test, these conditions will give rise to a curve similar to that of a single component giving two phases due to variable interaction with solvent (for the latter see Northrop et al., 1948). In his variable solvent solubility studies of P-lactoglobulins A and B, Tombs (1957a,b) obtained evidence of two invariant breaks for 0-lactoglobulin A and one variable transition point. The latter was interpreted as being due to an impurity present in his sample and he presented evidence to show that it was partly removed on fractionation. He does not appear to have considered that the invariant breaks could have arisen from polymerization reactions of the p-lactoglobulin. Providing the conditions given above are obeyed, one invariant break could arise from the monomer dimer equilibrium and the other from the dimer octamer equilibrium. Tombs’ tests were carried out in ammonium sulfate-sodium acetateacetic acid at pH 4.9, varying the ammonium sulfate concentration, conditions under which the (‘octamerization” reaction could possibly occur. Tombs’ curves for p-lactoglobulin B are not so well defined. They exhibit one definite invariant transition point and possibly a second one, and there may be two variable points due to impurities in the p-lactoglobulin B. Fractionation did not give prot.ein that obeyed the constant solvent solubility test for a ‘(pure” protein, but fractionation of the A variant did (cf. the data of Treece et al., 1964, in dilute NaCl). It would be preferable to carry out solubility tests in the absence of acetate and at pH values removed from those where the octamerization reaction can occur. Better methods are now available for purifying the &lactoglobulins and it would be interesting to repeat variable solvent solubility tests on them. If monomer and dimer are capable of existing in separate solid phases together, invariant points due to this could possibly be found from studies in a variety of solvents. Use could possibly be made of this in structure studies (see below).

+

180

H. A. MCKENZIE

4 , X-ray Crystal Structure Studies of b-Lacloglobulin Green et al. (1956) determined the unit cell dimensions of crystals of the bovine A and B variants. The A variant forms rectangular (orthorhombic) crystals (type Q). The B variant can exist in a metastable monoclinic form (type R) and in a stable orthorhombic form. There are small differences in the unit cell dimensions of the orthorhombic forms of the variants, Aschaffenburg et aE. (1965) found buffalo P-lactoglobulin crystals to be isomorphous with those of bovine 8-B type R. Green and Aschaffenburg (1959) prepared a cadmium derivative of bovine P-lactoglobulin B, following Prdaux at al. (1954) and Lontie and Prbaux (1955). They also prepared a pchloromercuribenzoate derivative of bovine P-lactoglobulin A. They showed, by X-ray diffraction studies, that in each case the molecule is composed of two identical units related by a twofold axis of symmetry. An idealized picture results of two spheres 17.9 A in radius impinging on each other by 2.3 A a t their surface of contact, giving a, center-to-center distance of 33.5 A. The molecular weight of the dimer unit is 36,000 and the radius of gyration is 21.7 A, in excellent agreement with that found for solutions at pH 5.7 by Wits et al. (1964) by small angle X-ray scattering. In a preliminary report Phillips (1963) cites Green and co-workers as having concluded, from the radial distribution of intensities from a salt-free crystal, that 8-lactoglobulin has some helical folding, and that the proportion of this may be as high as that of hemoglobin. Subsequent work appears to have made this conclusion doubtful. The work of Green and his group on the detailed structure of ,8-lactoglobulin is well advanced but there are a number of formidable problems. Aschaffenburg et al. (1965) have prepared crystals of p-lactoglobulin from ammonium sulfate solution. A triclinic type (X) was obtained below pH 6.9, whereas above pH 7 two crystal types (Y and Z) formed. Types Y and Z crystals themselves appear to contain identical molecules that are a conformational variant distinct from type X. Both the Y and Z crystals have a single unit of molecular weight 18,000 as the asymmetric unit of the crystal. Aschaffenburg et al. discuss whether or not the dimer molecule is dissociated into the monomer under the conditions of crystallization, and discuss possible amino acid residues involved in the area of contact between the units. They point out that the Y and 2 crystals are favorable for detailed study because of the 18.000 asymmetric unit, and have proceeded to study suitable heavy metal derivatives. They are aware that these crystals are formed in a pH region where &lactoglobulin is known to undergo a conformational change in solution (as has been discussed in Section VI). Structural studies of such derivatives must result in a structure that is not

MILK PROTEINS

181

applicable to the protein near its isoionic pH. It is the reviewer’s opinion that variable solvent solubility studies (see subsection 3 above) should be undertaken in a variety of solvents in the pH range 3.5-5.4. The solid phases on either side of any points of invariance should be examined by X-ray methods. It may be possible (subject to the conditions of Section VI,C,3) to find media from which crystals from monomer and/or dimer and/or octamer may be obtained. 6. Amino Acid Composition of P-Lactoglobulins. The first work on the amino acid composition of genetic variants of p-lactoglobulin was that of Piez et al. (1961) and Gordon et al. (1961) on bovine p-lactoglobulins A and B. Since this work, Bell et al. (1966b; see also McKenzie, 1965; Bell and McKenzie, 1964) have studied the amino acid composition of genetic variants of several species. Kalan et al. (1965) have examined the composition of bovine p-lactoglobulins A, B, and C, and Townend et al. (1965) have examined lactoglobulins A and B from milk samples of large numbers of cows. Also Phillips and Jenness (1965) have determined the amino acid composition of caprid /3-lactoglobulin. It is not easy to make an exact comparison of analyses from different laboratories, when there are such small differences in amino acid composition between the individual variants. However, a tabulation of the analyses from the various laboratories is made in Table VI-7. In making a comparison it is advisable to take cognizance of the following points: (1) All the analyses have been converted to a common basis, namely, an integral number of glycine residues. The mean analysis of each group of workers was converted in every case. (2) The analyses are expressed in terms of the number of residues per monomer unit. This has a molecular weight of 18,000 f 500. (3) Amino acid residues have been grouped in the same way as for the caseins (Section V). (4) The cystine analyses are not correded for loss in hydrolysis. In the case of serine and threonine, most workers have corrected their analysis for losses. (5) The analyses by Piez et al. were performed on A and B variants separated from pooled @-lactoglobulin. The others analyzed samples isolated from the milk of single homozygous animals. The results of the various groups are generally in good agreement. In Table VI-8 the reviewer has given what he considers to be the most likely values for the amino acid composition of the variants. The values for cystine in this table have been corrected, taking into account the total sulfur, methionine, and cysteine contents. The latter were obtained from the work of Leslie et al. (1962) and Phillips and Jenness (1965). Differences between the number of amino acid residues per monomer

TABLE VI-7 Amino Acid Composttion of &Ladoglobulins: Comparison of Variuus Workers’Result8 (Residues per monomer M.W. 18,000) Species:

Bovine

Ovine

A

Variant:

A

B

C

Workers.:

(PDFG) (TBK) (KGW) (BMS)

(PDFG) (TBK) (KGW) (BMS)

(KGW) (BMS)

GlY Ala Ser

Thr pro Val Ileu Leu Phe Tyr

‘pry CYSP

Met ASP

Glu

N W h 3 i

His LYS

3 13.9 6.9 8.1 8.2 9.9 9.7 21.9 3.9 3.6 2.5 3.9 3.7 16.0 25.0 2.8 1.9 14.9

3 14.2 6.3 7.4 7.9 9.6 9.5 20.3 3.6 3.5 3.5 3.7 15.4 23.7 -

-

-

-

3 13.4 6.6 7.8 8.5 9.7 9.5 20.5 3.9 3.9 2.0 3.3 3.9 15.2 23.9 3.0 1.9 14.4

3 14.2 6.1 7.5 8.3 9.7 9.0 22.5 4.0 4.0 1.9 3.9 3.8 16.0 26.2 15.6 2.9 1.9 14.6

4 14.9 6.9 8.1 8.2 8.9 9.7 21.9 3.8 3.6 2.5 4.1 3.7 15.0 25.0

-

2.9 1.8 14.8

4 14.5 6.5 7.4 8.8 8.8 9.5 20.3 3.6 3.5

-

3.6 3.7 14.6 24.9

-

4 14.4 6.6 7.8 8.4 8.9 9.6 21.0 3.9 3.9 2.1 3.3 3.9 14.9 24.7

-

3.0 1.8 14.4

4 15.2 6.3 7.6 8.3 8.9 9.1 22.6 3.9 3.8 1.8 3.4 3.9 15.2 26.4 15.4 2.9 1.9 14.8

4 14.4 6.6 7.6 8.5 8.9 9.6 21.0 3.9 3.9 2.1 3.3 3.9 14.5 23.6

-

3.0 2.9 14.4

4 15.4 6.4 7.78.5 8.9 9.1 23.1 4.0 4.0 2.0 3.5 4.0 15.4 25.5 14.7 3.0 2.9 14.9

5 15.4 5.5 7.5 8.0 9.5 8.1 19.9 3.8 3.7 2.0 2.9 3.5 14.9 24.0 17.0 2.9 2.1 13.6

Caprid

B

5 15.7 5.2 7.5 7.9 9.7 8.3 20.4 3.8 2.9 2 .o 2.9 3.8 14.6 23.6 17.2 2.9 2.9 14.0

5 14.6 5.5 7.2 8.1 9.4 8.5 20.0 4.1 3.9

-

3.0 3.4 14.5 23.8 16.0 2.8 2.0 14.8

5 15 7 8 8 10 9 20 4 4 2 5 4 15 25 14 3 2 15

a PDFG = Piez, Davie, Folk and Gladner (1961); KGW = Kalan, Greenberg, and Walter (1965); TBK = Townend, Basch, and Kiddy (1965); BMS = Bell, McKenzie, and Shaw (1964, 1966b);PJ = Phillips and Jenness (1965). * Estimate from values obtained from amino acid analyzer.

183

MILK PROTEINS

unit of the individual variant and those of the bovine B variant are compared in Table VI-9. The bovine A variant differs from the bovine B variant by +1 Asp, +1 Val, - 1 Gly and - 1 Ala, whereas the C variant differs from B by 1 His and - 1 Glu (N). Also included are figures for the recently discovered beef cattle variant, p-lactOglobUlinDroust~~t~, (Dr), isolated by Bell et al. (19664. This

+

TABLEVI-8 Probable Number of Amino Acid Residues per Monomer of &L&qkibulin

GlY Ala Rer Thr Pro Val Ileu Leu Phe TYr Try Cystine Cysteinr Met ASP Glu

NHa"

Arg His LYS

3

14

7

8

8

4

15

7

8 8

9

4 15 8

5 15 6 8

5 15 6 8

10 9

10 9

20

20 4 3

7

8 9

10 10

10

10

22

22

4 4 2

4 4

22 4

2 1

4 16 25 15 3 2 15

2 2 1

4 15

4

2 2

4 15

25 15 3 2

24

15

15

14

3 3

8

4 4

2 2

4 15 24 16

3

2 14

8

2

2

4 15

24

16

3 3 14

5 15 6 8 8 10 9 20 4

4

2 2 1 4 15 24 15 3 2 15

variant has a number of amino acid residues identical to that of the bovine A variant but contains sialic acid and glucosamine. Mawal et al. (1965) obtained an amino acid analysis for buffalo p-lactoglobulin similar to that for the bovine B variant (cf. Sen and Sinha, 1961). Nui and Fraenkel-Conrat (1955) found two amino-terminal leucine residues and 2 carboxyl-terminal isoleucine residues per 36,000 molecular weight dimer of pooled 8-lactoglobulin. Identical results were obtained by Kalan et al. (1965) for bovine p-lactoglobulins A, By and C (see also Kalan and Greenberg, 1961). The amino acid carboxyl-terminal sequences

184

H. A . MCKENZIE

appeared to be identical. Thus the amino acid substitutions in the bovine variants do not occur a t or near the ends of the chains. Kalan et al. (1962) located the peptides containing one of the substitutions (Asp/Gly) between the bovine A and B variants. The environment of this substitution has been examined by Townend (1965), and his results considered in relationship to the octamerization reaction (vide infra). These findings have been confirmed essentially by Bell et al. (1966b). TABLE VI-9 Differences in Number of Amino Acid Residues per Monomer Compared with Bovine B Species: Variant:

Residue GlY Ala Ser Val Ileu Leu TYr ASP

Glu His Lys Hex" Siab

Bovine

Ovine

Caprid

B

A +1

+1

-1

-1 +1

-

+1 -1 -2

-

-1 -2

-

-1

-1

-1

-

-1 -

-

+1

-1

-

Hexosamine. Sialic acid.

The Val-Ala difference in bovine A and B does not appear to have been detected in peptide studies at Philadelphia or Canberra,. However, Pfuderer (1961) considers that he has found some evidence of it. Unfortunately this work does not appear to have been published. The amino acid analysis indicates that bovine C has 1 His and - 1 Glu(N) with respect to bovine B. The method does not enable a distinction to be made between the acid and the amide. If a single mutation is involved, both differences, His/GIu and His/Glu(N), are possible. Kalan et al. (1965) have argued from mobility differences in acrylamide electrophoresis that the difference is His/Glu(N). However, the situation on gel electrophoresis is not as simple as they imply, and in the reviewer's opinion this argument does not resolve the problem.

+

MILK PROTEINS

185

Bell et al. (1966b) have isolated the difference peptides from the tryptic cores of B and C . The two peptides have identical mobility a t pH 8.9, hence the histidine in C has replaced a glutamine in B and not a glutamic acid residue. Greenberg and Kalan (1965) have prepared modified bovine A, B, and C proteins by the action of carboxypeptidase. They concluded that the C-terminal isoleucine and penultimate histidine are located on the external surfaces of the molecules. Comparison of peptide maps of bovine A and Droughtmaster shows an obvious difference in mobilities of one arginine-containing peptide; amino acid analyses of this indicate that they have the same composition but that Droughtmaster contains, in addition to amino acid residues, covaleritly linked glucosamine and sialic acid (B.M.S.). Bell et al. (196613) have shown that the ovine B variant has - 1 Tyr and +1 His with respect to the A variant. The His difference has been related to a Glu [Glu(N)] residue and hence is not a simple substitution for Tyr. The B variant appears to contain some sialic acid. While the amino acid compositions of the ovine and caprid variants resemble those of the bovine variants, there are considerable differences between the three species, as shown in Table VI-9. The biosynthetic significance of the genetic variants of milk proteins has been reviewed by Larson (1965). 6. Immunological Properties of @-Lactoglobulins

Larson and Twarog (1961) found that bovine @-lactoglobulinsA and B were weak antigens compared with serum proteins, and that they were immunologically similar (see also Gough and Jenness, 1962b). Bell and McKenzie (1964) produced antisera against bovine p-lactoglobulin AB and p-lactoglobulin C in rabbits. The j3-AB antiserum gave a single precipitin line with all three proteins by the Ouchterlony double-diffusion technique. On immunoelectrophoresis a single precipitin arc was obtained with @-A, @-B and p-C against the 8-AB antiserum. Similar results were obtained when the proteins were mixed two a t a time (1:l) and subjected to immunoelectrophoresis. The experiments were repeated with 0-C antiserum. All the results indicated that bovine p-A, 8-B and 8-C are immunologically identical. By similar methods it was found that ovine &A and P-B are immunologically identical. Furthermore, the ovine &lactoglobulins reacted strongly with complete cross-reaction with antiserum to the bovine proteins. The authors concluded that either the antigenic portions of each of the bovine

186

H. A. MCKENZIE

and ovine @-lactoglobulinsare identical and the difference amino acid residues are not involved in them, or the rabbit is incapable of detecting the differencesbetween the two molecules and producing specific antiserum. Subsequently Larson and Hageman (1965) showed from quantitative immunological tests by the Oudin procedure that the bovine A, B, and C variants are identical. However, they confirmed the earlier work of Johke et al. (1964) that there are small quantitative differences between species (ovine, bovine, and caprid). With dilute samples they obtained multiplicity of lines. One cannot help but wonder whether this involves dissociation of the p-lactoglobulin. 7. Summarg of p-Lactoglobulins

The p-IactoglobuIins provide a fascinating example of biochemical evolution. Small changes in amino acid composition give rise to differences in their properties. Aschaffenburg and Drewry first found evidence of genetic polymorphism in the bovine P-lactoglobulins with their discovery of the A and B variants in European dairy breeds. Later Bell discovered the C variant, so far appearing to occur only in the milk of Jersey cattle. More recently Bell, McKenzie, Murphy and Shaw isolated a new variant 8-lactoglobulinu,,,,htmaater from this species of beef cattle. Bell and McKenzie isolated two variants (A and B) from ovine milk. There appears to be only one variant in caprid milk. By contrast, there is no p-lactoglobulin in human milk. Studies are in progress in several laboratories on porcine, equine, kangaroo, and echidna milk. It may be anticipated that interesting behavior will be observed in the whey proteins of these species. Of all the p-lactoglobulins discovered so far, only bovine 8-lactoglobulin A undergoes octamerization a t pH 4.6 and low temperature. This property appears to be associated with the one extra aspartic acid residue per monomer of this molecule. The tendency to dissociate of the dimer form of 8-lactoglobulins, the predominant form a t moderate concentration near the isoionic point, depends on the pH, temperature, and ionic strength. The order of dissociation of the variants depends on the pH. Near the isoionic point it is possible that all the p-lactoglobulins have a conformation consisting of helical, &like, and disordered structures. The conformation undergoes a change near pH 6 and above, and may result, especially a t the higher pH values, in irreversible changes, a t least some of which involve intermolecular polymerizations. The p-lactoglobulins provide excellent examples of the pitfalls that beset the investigator in the study of heterogeneity, associationdissociation, and conformation of proteins.

MILK PROTEINS

187

D. a - h h l b u m i n 1 . Introduction

Pedersen (1936) observed a slow moving peak in the sedimentation pattern of whey proteins, which he designated the “a-peak.” Kekwick (1936) isolated a crystalline protein that appeared to be responsible for the a-peak. Subsequently Svedberg (1937) found the molecular weight of this protein to be 17,600and called it a-lactalbumin. The protein was characterized more completely by Gordon and Semmett (1953; see also Gordon et al., 1954). Their isolation from acid whey was based on observations of Sorensen and Sorensen (1939) on the solubility in ammonium sulfate solutions. cu-Lactalbumin constitutes about 20 % of the whey protein (Larson and Jenness, 1955). The isolation of a-lactalbumin has been discussed generally (Section VI,A). It is important to note that some apparent heterogeneity is observed in most preparations of this protein. At present it is difficult to interpret unequivocally many of the heterogeneity experiments carried out on this protein. 2. Genetic Variants of a-Lactalbumin An investigation of milk whey proteins of European cattle by Aschaffenburg and Drewry (1955), using filter paper electrophoresis, revealed two genetic variants of p-lactoglobulin but only one a-lactalbumin. Subsequently Blumberg and Tombs (1958) carried out a similar examination of whey proteins from milk of Icelandic and Nigerian cattle. They observed similar genetic polymorphism of &lactoglobulin to that observed by Aschaffenburg and Drewry for the European cattle. In samples of milk from Nigerian cattle of Zebu origin (White Fulani), there occurred one a-lactalbumin band similar in mobility to the band in European breeds, or a band of higher mobility, or both bands occurred together. They considered this to be an example of genetic polymorphism, and designated the faster moving a-lactalbumin of the Zebu as a-lactalbumin A, and the slower moving protein common to the Zebu and European breeds as cy-hctalbumin B. Bhattacharya et al. (1963) examined Indian Zebu and buffalo for genetic polymorphism of a-lactalbumin and p-lactoglobulin, using filter paper electrophoresis. They found that the A and B variants of both proteins could occur in the Zebu and that the observed and expected frequencies from the Hardy-Weinberg law were in close agreement. Buffalo milk contained a /3-lactoglobulin having a mobility similar to that of European bovine p-lactoglobulin B, and an a-lactalbumin with mobility similar to that of the Zebu a-lactalbumin A.

188

H. A. MCKENZIE

Work by Bell and McKenzie (1964, 1966a,b,c), using starch geI and filter-paper zone electrophoretic methods, revealed only a-lactalbumin B in dairy cow milk whey samples, but the presence of either or both a-lactalbumins in the milk of Droughtmaster beef cattle. They found only one a-lactalbumin in sheep and goat milk. The latter observation is in accord with the work of Sen and Chaudhuri (1962). Although no p-Iactoglobulin has been detected in human milk, a-lactalbumin is present (Bell and McKenzie, 1964; Johansson, 1958; Maeno and Kiyosawa, 1962). 3. Some Observations on Heterogeneity of a-Ladalbumin

Gordon and Semmett (1953) found their preparation of a-lactalbumin to give normal diffusion and sedimentation curves at pH 7.5 (0.02 I PO4, 0.18 I NaCI). The sedimentation measurements a t pH 8 and 28’ gave szo,w = 1.75 S, and diffusion measurements gave Dzo,, = 10.57 F. Using Groves’ value of 0.735 for B, they obtained an Mappof 15,100. They obtained a single peak in moving-boundary electrophoretic experiments at pH 2 and 3.0 (glycine-HC1 buffer), pH 6.6 and 7.7 (POsbuffer), and pH 8.5 (veronal buffer). However, in lactate buffer at pH 3.3 they observed two peaks. Zittle and DellaMonica (1955) found that Gordon and Semmett’s preparation was precipitated completely by 3.0 M ammonium sulfate. Extraction of the precipitate with 2.5 M ammonium sulfate gave a solution of a soluble form of a-lactalbumin (soluble in 2 M (NH4)804). When the remainder of the precipitate was extracted with 1.5 M ammonium sulfate, a solution of an “insoluble form” (insolublein 2 M (NH4),S04)was obtained. This transformation has been thoroughly investigated by Zittle (1956). Dialysis of the total preparation against water produced about 75 % of the “insoluble form.” However, dissolution of the crystalline precipitate in water (i-e.,dilute (NH4)zS04)or 0.1 M sodium chloride gave 95 % of the “soluble form.” The addition of sodium chloride to a-lactalbumin at pH 6.6, giving a concentration of 0.17 M in sodium chloride, produced an immediate shift in pH to 6.1, and a slow transformation as measured by the solubility in 2 M ammonium sulfate. The effect was similar when sodium chloride was added to the protein solution a t pH 8.8. There appeared to be no difference in the moving-boundary electrophoretic and sedimentation properties of the 2 M ammonium, sulfate “soluble” and “insoluble” forms. The solubility transformation was also effected by a variety of other salts. There was no change following the transformation in the value of [ C Y ] ~ ~ of S -60”. When they took an aqueous a-lactalbumin solution a t pH 5.9 and added

MILK PROTEINS

189

sodium chloride to it to 0.1 M , there was an immediate drop in pH to 5.4. A precipitate slowly formed in the 0.1 M NaCl solution consisting of about 30 % of the a-lactalbumin. This precipitation is not related directly to the “solubility transformation.” It arises because the binding of C1shifts the pH of the a-lactalbumin solution into the pH region where its solubility is low. If a similar experiment is carried out a t pH 5.9 but the solution is then made 0.5 M in sodium chloride, the pH drops to 3.6. There is no loss in solubility, as the pH is outside the pH zone of low solubility. The binding of anions outside this pH region results in transformation of a t least part of the a-lactalbumin to a form that becomes insoluble in 2 M ammonium sulfate. Zittle concluded that this solubility test is possible because the “insoluble” form is completely precipitated under these conditions and has no chance to revert to the “soluble” form. Klostergaard and Pasternak (1957) examined the patterns of a Gordon and Semmett-type preparation of a-lactalbumin in moving-boundary electrophoretic and sedimentation experiments. At a concentration of ca. 10 gm/liter, they obtained bimodal descending electrophoretic patterns for pH 3.3 (lactate buffer), 7.5 (phosphate), 7.5 (Tris), and 8.5 (veronal). The ascending and descending patterns were highly nonenantiographic. Patterns at pH 3.3 ( I 0.1, lactate) were protein concentration-dependent. An extended experiment (16 hours, 10 V cm-*) for a protein concentration of 15.5 gm/liter did not result in complete resolution of the two peaks of the bimodal pattern. Re-electrophoresis of material corresponding to the fast peak again gave a bimodal pattern. It was shown in other electrophoresis experiments that P-lactoglobulin was not a contaminant. Sedimentation measurements at pH 3.3 and 7” gave a single peak with some boundary spreading a t 15 gm/liter protein and an s20,w= 2.0 S. They found from electrophoresis mobility measurements that the isoelectric point was 5.1, which is removed from the region of minimum solubility (4.14.8). Wetlaufer (1961) examined a number of samples of a-lactalbumin prepared by the method of Gordon and Semmett. He measured szO.,,. in the concentration range 5-15 gm/liter a t pH 6.0 ( I 0.2, P04-CI) and obtained a small negative slope for his s vs. C curve with an extrapolated S O Z O , ~ of 1.87 S. In moving-boundary electrophoretic measurements at pH 7.5 he found 5-30 % of a fast moving peak in different preparations. (A similar result was obtained on filter paper electrophoresis a t pH 7.5.) A sample of the fast peak removed from the moving-boundary electrophoretic cell . ~ 1.80 S (at 5 gm/liter) and an ultraviolet spectrum similar gave an ~ 2 0 of to that of the original a-lactalbumin. Kronman and Andreotti (1964) examined samples prepared by the Gordon and Semmett (1953), Aschaffenburg and Drewry (1957a), and

190

H. A. MCKENZIE

Robbins and Kronman (1964) methods (see Section V1,A). All samples gave a single symmetrical peak in sedimentation experiments at pH 8.6. A fast moving, trace band was observed in starch gel electrophoresis at alkaline pH. This band was reminiscent of the fast moving peak observed in the descending pattern moving-boundary electrophoresisand filter-paper electrophoresis. Now in gels containing 5 M urea at pH 3.0 (formate buffer) a “trace” band was observed moving behind the main band. Taking all the above results together, the reviewer is inclined to the view that the bimodality of the moving-boundary electrophoretic patterns and the minor band in zone electrophoresis arise from a reaction involving complexing between a-lactalbumin and a buffer component. Also it does not seem that polymerization of the a-lactalbumin plays a major role in the patterns reported. However, recent work of Kronman and his collaborators indicates thzt the association and aggregation processes of a-lac talbumin in acid solution are strongly dependent on pH and time. It would be desirable to repeat some of the electrophoretic experiments in the light of important factors revealed by their study (which is discussed in Section VI,D,3). Several puzzling observatipns have been reported recently. SzuchetDerechin and Johnson (1965a) presented patterns from electrophoresis in starch gel a t p H 9.0 (borate) and on filter paper at pH 8.6 (veronal) of their preparations of a-lactalbumin. They observed only a single band. This a-lactalbumin wm prepared by DEAEcellulose chromatography (see Section V1,E). Groves (1965) has also developed a method for the preparation involving DEAE-cellulose chromatography. He made an electrophoretic examination of a rechromatographed fraction, which even on rechromatography gives an asymmetrical pattern with a trailing zone reminiscent of a reaction boundary. Part of this fraction gave a single band on zone electrophoresis. He was unable to produce the fast minor band, even after exposing the preparation to conditions similar to those used in the older fractionation procedures. These observations warrant further study. Such a study is in progress in the reviewer’s laboratory.

4. Molecular Size of a-Lactalbumin A careful study of the effect of pH on the molecular size of a-lactalbumin has been made by Kronman and Andreotti (1964) and Kronman et al. (1964). At pH 8.5 (0.15 M KCl) their preparations of a-lactalbumin (see above) gave a single peak in the sedimentation velocity pattern. They present no patterns for this pH value in their paper, but state, “A very small amount of heavy material was noted as a slight thickening of the pattern

25t l5

35

t

t

10 0

0.2

0.6

0.4

0.8

grn/ 100 rnl I100

(0)

25

0.2

I

0.45

06

L

0

gm/100 ml (C)

FIG.VI-19. Plots of apparent weight average molecular weight vs. concentration for a-lactalbumin: (a) pH 8.55 (0.15 M KCl); (b) pH 2.00 (0.15 M KCl): 0 , 10'; 0 , 25'; (c) pH 3.00 (0.15 M KCI): 0 , 10'; 0, 25'. (After Kronman and Andreotti, 1964.)

(arn)

I 5 t

1

10 0

0.8

02

Q4

Q6 gm / 100 ml (b)

a8

1.0

192

H. A. MCKENZIE

FIG. VI-20. (a) Sedimentation patterns showing effect of time of exposure on polymerization of rr-lactalbumin at 25" (speed: 59,780 rpm, phase plate angle: 60",time: 48 min after reaching speed): pH 2.00 (0.15 M KCI) solution measured in 4 mm-Dural cell, pH 3.00 (0.15 M KCl) solution measured in 12 mm-KelF cell. (b) Concentration dependence of the sedimentation coefficient (s~,,,,.) of the slow peak for a-lactalbumin at pH 3.00: - - - - -, experimental values corrected for concentration dependence; .., data for pH 8.6. (After Kronman and Andreotti, 1964.)

...

193

MILK PROTEINS

3.0 2.5

I

1.51 I.o

0

1

2

3

4

5

6

7

gm/IOOml

(b)

FIG.VI-20 (cont.).

on the fast side of the boundary." They obtained the following concentration dependence for the sedimentation coefficient ( S Z ~ , ~ ) , ~ z 0 . w = 1.92 + 0.02 - (6.5 k 1.0)X 10-8 C where C is the protein concentration (in gm/liter). Sedimentation equilibrium measurements under the same pH conditions gave the results presented in Fig. VI-19. It will be noted that the apparent weight average molecular weight obtained by extrapolation is 16,200 in good agreement with the value of 16,300 obtained by Wetlaufer (1961) from osmotic pressure studies, and of 15,500 obtained by Gordon vs. C curve and Ziegler (1955) from amino acid analysis. Also the i@w,app has a positive slope. This could arise from association and/or the sign of the second virial coefficient. If they titrated cw-lactalbumin solutions to pH 2.0 and pH 3.0 (concentrations 58 and 15 gm/liter, respectively) and t#hencarried out sedimentation velocity measurements 1 hour and 24 hours after mixing, they obtained the sedimentation patterns shown in Fig. VI-20. It can be seen that the slow moving peak (ca. 1.95) is asymmetrical and that increasing amounts of a fast moving peak (szo= 18 S) appear a t both pH values with increasing time. At 24 hours considerable resolution of the two peaks is obtained. At lower concentrations they observed only a single peak a t pH values of 2.0 and 3.0. The concentration dependence of the slow moving peak is

194

H. A. MCKENZIE

shown in Fig. VI-20, and the concentration dependence of in Fig. VI-19. Sedimentation patterns a t pH 5.24 (59 gm/liter, lo") indicated a single peak, those at pH 6.0 (81.6 gm/liter, 10' and 25") showed considerable fast moving material distorting the fast moving side of the peak. There was a small decrease in s20.w with increasing C at both pH values, and a value of 2.0 S was obtained for S'ZO,~. The value of il?w.app increased very slightly with increasing concentration. Thus Kronman and Andreotti concluded that there is no strong tendency to association of a-lactalbumin at pH 5-6 in 0.15 M KCl. Considering all these results together, one thinks immediately that a slow association of a-lactalbumin is involved on the acid side of the isoelectric point, the slow moving peak in the sedimentation patterns consisting of monomer and a small amount of dimer, trimer, etc., and the fast moving peak consisting of highly polymerized a-lactalbumin. Kronman and Andreotti prefer, however, another explanation, since they conclude that a more complex situation exists. They consider the concentration dependence of s20,wfor the slow peak to indicate that there is an association of the rapidly re-equilibrating type. Also they consider the time dependence of the appearance of the fast moving peak to indicate a timedependent polymerization, leading to a high degree of polymerization superimposed on the rapid association involving only a monomer-dimer association. Some support for this interpretation is obtained from the temperature dependence of the low pH association leading to the slow peak and the polymerization leading to the fast peak. The former appears to increase with decreasing temperature, the latter to increase with increasing temperature. On the other hand, their explanation demands that monomer and dimer exist with polymers of large size without polymers of intermediate size. It is puzzling why this should be so. Kronman et al. (1964) found that the time-dependent polymerization at low pH leads to a fast moving peak with an szo,wof 10-18 S in contrast with the value of 1.93 S for the monomer. The rate of formation of the heavy components decreased with decreasing pH, ionic strength, and temperature. Reduction of the net molecular charge through anion binding increased considerably the rate of aggregation. No highly polymerized forms of a-lactalbumin were found on the alkaline side of the isoelectric point. The low pH polymerization could be readily reversed by adjusting the pH value of such solutions or gels to pH values higher than the isoelectric pH (5.1). Raising the pH above 3.8 resulted surprisingly in a decrease in the rate of polymerization (contrary to simple electrostatic considerations). Kronman and his co-workers conclude that at low pH (<4) the a-lactalbumin molecule undergoes a conformational

195

MILK PROTEINS

change that results in a form of the molecule having a greater tendency to polymeriBation and a lower solubility than the native molecule. 6. Conformation of a-Lactalbumin

Before considering the conformational changes of a-lactalbumin it is of interest to note the amino acid composition obtained by Gordon and Ziegler (1955), shown in Table VI-10. The tryptophan content is high, but the overall frequency of nonpolar side chains is not unduly high, having a value of 0.32 (by the definition of Waugh, 1954). TABLE VI-10 Amino Acid Composition of Bovine rY-lactalbuminn (Residues per monomer M.W. 15,554;total N(%) = 15.86) GlY Ala Ser

Thr pro Val Ileu Leu Phe Tyr

6.6 3.7 7.0 7.2 2.1 6.2

8.1 13.7 4.2

Try CYS/2 Met ASP Glu N Hz Arg

His LYS

5.3 4.1 1 .o 21.8 13.6 15.2 1 .o 2.9 12.2

4.6

After Gordon and Ziegler (1955).

Kronman et al. (1965) investigated the possibility of a conformational change in a-lactalbumin a t low pH values by the technique of difference ultraviolet spectra. They found a hypsochromic (“blue”) shift in the ultraviolet spectrum at pH values below 4. The difference spectrum showed a “peak” a t 287 and 293 mp (AA was negative with respect to the native protein a t pH 6). The difference a t 293 mp in other proteins is known to arise from differences in the environment of the tryptophan groups. It was found that AApgafor a-lactalbumin depends strongly on the pH but is insensitive to changes in ionic strength. I n the pH region 3 4 , AAzea depends strongly on the temperature. None of the spectral changes appeared to be completely reversible. The difference spectrum was abolished in 8 M urea (protein in 8 M urea a t pH 3.4 vs. protein in 8 M urea a t pH 6). Thus the generation of the difference spectra for a-lactalbumin a t low pH requires the existence of the characteristic conformation of thp native protein in the reference solution. The electrostatic interaction factor w appeared to decrease a t low pH. Kronman and Holmes (1965) examined the degree of exposure of the tryptophan groups of a-lactalbumin under a variety of conditions, using

196

H. A. MCKENZIE

the solvent perturbation method of difference ultraviolet spectrophotometry (Herskovits and Laskowski, 1962). The native protein a t 25” has only two tryptophan groups ((exposed”to the perturbants DzO, sucrose, ethylene glycol, and glycerol, both at pH values above the isoelectric point and a t low pH (1.8-3). On the other hand, all five tryptophan groups are exposed following reduction of the S-S bonds of the protein, while in 8 M urea four of the tryptophan residues are exposed. The absence of a change in number of tryptophan groups exposed with respect to any of the perturbants, in going from pH 6 to p H 1.8 a t 25”) demonstrates that the hypsochromic shift in ultraviolet spectrum observed above cannot be the consequence of an unfolding of the moIecule in the neighborhood of the t.ryptophan groups. They found, on lowering the temperature to 1’ at pH 6, that two groups that are exposed a t 25’ became “buried” with respect to the large perturbants, but remained accessible to DzO. This “crevice” closure is more limited at pH 1.8-3.0: one group is buried with respect to sucrose and glycerol, but two are accessible to DzO. The data reported by Herskovits and Mescanti (1965) on the optical rotatory properties of a-lactalbumin indicate values of a0 and bo a t pH 7.0 ( I 0.1) of -355 and -235, respectively. In 8 M urea a t pH 3.2 they found values of -710 and -10 for a. and bo, respectively. From these limited data it appears that native a-lactalbumin has a fair content of a-helix. 6. Summary of a-Lactalbumin

The second most abundant milk whey protein, a-lactalbumin, shows interesting properties of ion binding, association, polymerization and solubility. The tendency to these reactions appears more marked on the acid side of pH 5 than on the alkaline side. The low molecular weight of 16,000 and the posvibility of isolation of genetic polymorphs make this a fascinating protein for further study. Surprisingly, it has received little attention. The observations of Kronman et al. on difference ultraviolet spectra for a-lactalbumin indicate that facile interpretation of difference spectra of proteins is dangerous without concurrent solvent perturbation difference spectral measurements.

I?, Iron-Binding Proteins 1. Introduction

Sgrensen and Sgrensen. (1939) first noted two colored proteins, one red and one green, among their bovine whey protein fractions. Evidence has accumulated in the last twenty-five years to show that these are ironbinding proteins. The red proteins play a role in iron nutrition and possibly in the defensemechanisms of the body, as they are capable of attracting

MILK PROTEINS

197

iron away from pathogenic bacteria and detoxifying exotoxins and endotoxins. The green protein is lactoperoxidase, the predominant enzyme in milk, sometimes rising to as much as 1 % of the total whey protein. Immunological studies of milk have enabled several groups of investigators (da Silva and Monteiro, 1959; Gugler el al., 1959;Hanson, 1960;Gahne, 1961) to conclude that there are two classes of red protein in human and bovine milk: one is specific to milk and is presumably synthesized in the mammary gland, the second appears to be identical with blood serum transferrin. I n the early sixties, several groups of workers in the United States (Groves, 1960; Gordon et al., 1962), Great Britain (Szuchet-Derechin and Johnson, 1962), Switzerland (Blanc and Isliker, 1961), France (Montreuil et al., 1960b), Germany (Gruttner et al., 1960), and Sweden (Johansson, 1960) reported the isolation of red proteins from either human or bovine milk. In this work, carried out more or less concurrently, various methods were used in the isolation of the proteins and different techniques in their characterization. Thus it is not always easy to make a direct and simple comparison between the work of the various groups. The proteins isolated were given various names: “iron-binding protein,” “red protein,” “ lactotransferrin,” ‘‘ iactosiderophilin.” The reviewer proposes the following nomenclature (see Section 111) and uses it in this article, although the term for a given protein may not always be that used by each investigator of it: Red-colored, iron-containing protein, specific to milk : Zacfoferrin. The iron-free form of lactoferrin: apo-lactofewin. Red-colored, iron-containing protein of serum : serum trunsferrin. When the serum transferrin sample has been isolated from milk, this is shown as a suffix in parentheses: serum transferrin (milk). The species from which a red protein has been derived is used as a prefix, e.g., bovine lactoferrin. 2. Isolation of Lactoferrin and Serum Transferrin from Milk

In the course of isolation of lactoperoxidase from bovine milk by column chromatography of whey protein fractions, Polis and Shmukler (1953) isolated a red protein in partially purified form. Groves (1960) isolated bovine lactoferrin from acid casein by extraction with 1 M acetic acid a t pH 4.0. It was then fractionated with ammonium sulfate and by DEAE-cellulose chromatography. Gordon et al. (1962) isolated it from rennet whey. Szuchet-Derechin and Johnson (1962, 1965a,b) made a careful study of the fractionation of bovine lactoferrin and serum transferrin (milk) from sodium sulfate whey. Groves (1965) isolated both types of protein from acid bovine whey. In a preliminary study, Szuchet-Derechin and Johnson (1965a) carried out DEAE-cellulose chromatography on bovine whey proteins, obtaining

198

H . A . MCKENZIE

50

Tube number

FIG.VI-21 (a). See legend, facing page.

seven fractions (see Fig. VI-2la), which they identified by filter-paper, starch gel, and (in some cases) immunoelectrophoresis as follows: F1 Lactoferrin primarily F2 Serum transferrin (milk) primarily F3 rrLactalbumin and other proteins F4 a-Lactalbumin F5 Serum albumin, plus a-lactalbumin, 8-lactoglobulin, etc. F6, F7 A mixture of proteins, mainly 8-lactoglobulin

MILK PROTEINS

199

FIG.VI-2l(b) FIG.VI-2la. Szuchet-Derechin and Johnson (1962-1966) method for isolating iron bonding proteins from sodium sulfate total whey proteins. Top: Fractionation of total protein by gradient elution from a DEAE cellulose column. Fractions 1-7 are Fl-F7 in text. Bottom: Fractionation, by stepwise elution from a DEAE cellulose column of a batch fraction (supernatant from DEAE cellulose, pH 7) of iron binding proteins (approximately equivalent to F1 and F2 above) from total whey proteins. FIG.V I S l b . Groves (1965) method of fractionation of total acid whey proteins for lactoferrin and serum transferrin. T o p : Elution pattern of whey proteins from a DEAE cellulose column. Fraction 3F was then chromatographed on phosphocellulose to give the serum transferrins. Bottom: Gel electrophoretic comparisons (pH 9.5) of transferrins (indicated by vertical arrows) found in milk and blood of individual cows. (a) Cow A, blood serum; (b) Cow A, whey fraction; (c) Cow B, blood serum; (d) Cow B, whey fraction.

(reject) N

0

0

(1M H2SO4 to pH 4)

I PPt. (reject)

(Rivanol 0.31 gm/lOO ml, pH 7.9)

casein (reject) - 1

super. (toPH 8

(NH&SOI

(Activated G, 10 hr, OD, centrifuge)

0.8 satd.) I

PPt.

(dialyze

0.02 M PO4, pH 8, DEAEcellulose column) Lactoferrin

I

super. (reject)

-1 residue

super. (25% EtOH pH 6.8, -So)

I-

FIG.VI-22. Isolation of lactoferrin from human milk.

(sat. (N%)asO* PH7) 7 super. PPt. (reject) (acid to pH 3.8) I

I

PPt.

(dissolve in Na cit. pH 5.2, Amberlite XE-64 column) Lactof errin

I

super. (reject)

MILK PROTEINS

20 1

Using this preliminary study as a basis, Szuchet-Derechin and Johnson (1965b) developed a batch chromatographic procedure giving a fraction rich in the iron-binding proteins. This fraction was subjected to column chromatography on DEAE-cellulose, giving two fractions F1 and F2, rich in lactoferrin and bovine serum transferrin (milk), respectively (see Fig. VI-21a). Szuchet-Derechin and Johnson (1966a) then chromatographed fraction Fl on a DEAE-cellulose column near pH 9. They applied F1 to the column a t pH 9.38 and carried out a stepwise elution near pH 8.6. They obtained six fractions, all containing iron-binding protein. Four of the fractions chromatographed similarly in repeated fractionation. On the other hand, the remaining two fractions consisted of protein modified from the original protein during the fractionation. The method, developed by Groves (1965) for bovine iron-binding proteins involving chromatography of an acid whey on a DEAE-cellulose column (see Fig. VI-2lb), gave in the preliminary chromatography a t pH 8.2, four fractions: 1F 2F 3F 4F

Greenish red in color: lactoferrin, lactoperoxidase, and other proteins Light red in color: lactoferrin Serum transferrin (milk) Mainly a-lactalbumin

Fraction 2F was used as a source of lactoferrin without further purification (checked by acrylamide electrophoresis). Fraction 3F was applied to a phosphocellulose column at pH 6.0 and eluted with a stepwise pH gradient (6-7) to yield bovine serum transferrin (milk). It was identified with transferrins prepared from serum of the same cow by acrylamide gel electrophoresis (see Fig. VI-2lb). The methods developed by Johansson (1960), Gruttner et al. (1960), Montreuil et al. (1960b), and Blanc and Isliker (1961) for isolation of ironbinding protein from human milk all appear to give lactoferrin and are outlined in Fig. VI-22. Each of the proteins was found by immunoelectrophoresis to be specific to milk. However, Blanc and Isliker obtained an additional fraction containing a &globulin similar to one present in the blood serum. They did not investigate it further. 3. Comparison of Properties of Iron-Binding Proteins

A comparison is made in Table VI-11 of some of the properties of ironbinding proteins of milk and serum transferrin. Amino acid compositions are compared in Table VI-12. In making this comparison, there are a number of points that should be made clear: (1) The isolated proteins designated as lactoferrins in the tables have all been shown by immunoelectrophoretic measurements to be milk

TABLE VI-11 Comparison of Properties of Iron-Binding Proteinsn Bovine milk

Source :

Human milk

Worker :

(GI

(S)

Serum transferrin (S, Ga, G )

Property Immunoe!ectrophoresis

-

Not similar t o S.T. 0.0

Similar to S.T. -

Lactoferrin

Protein:

Electrophoretic mobility, pH 8 (lo+ cm2V-’ See-1) Molecular weight Sedimentation coeff. (sz0.w) (8) p H of dissociation

Fe

(%I

Fe atoms/mole Absorption max. (mp) N (%) Total carbohydrate (%) Hexose (%) Hexosamine (%) Sialic acid (%)

~~

~~

0.0

86,000 5.5(c = 0)

93,000 f 3000 5,65(c = 0) 5(c = 0)

2

-

2 470 14.84 7.2 4.54 2.21 0.3

2 453-470

0.11

-

-

Lactoferrin

(J) Not similar t o S.T.

-

-

-

-

2 0.13-0.15

460

460-470

-

Human blood

-

(B)

(MI

Not similar t o S.T. -3.5

88,OOO 4.3(c

=

2

1)

0.11 2 400455 7.2 4.1 2.3 0.8

Not similar to S.T. -3.45 89,OOo-95,000 4.7(c = 0) 1.75 0.35 at sstn. 6 452 14.84 7.17 3.9 2.4 0.87

Serum transSerum ferrin transferrin (B,H, Si) (Sc) Similar t o S.T.

-

-

-3.1

-

6 . l ( c = 0)

-

-

-

-

90,OofJ 4-6

2 470 15.4

5.5

2.4 1.6 1.4

~

a S.T.= Serum transferrin from blood. G = Groves (1960, 1965); S = Szuchet-Derechin and Johnson (1965a, 1966a); Ga = Gahne, Rendel and Venge (1960); J = Johansson (1960); B = Blanc and Isliker (1961); hl = Montreuil, Tonnelat and Mullet (1960); Sc = Schultze, Heide and Miiller (1957); H = Hanson (1960); Si = da Silva and Montreiro (1959).

203

MILK PROTEINS

TABLE VI-12 Amino Acid Composition of Iron Binding Proteins (No. of residues per molecule) Source: Prot.ein: Workera : Mol. wt.: Residue

GlY Ala Ser Thr Pro Val Ileu Leu Phe Tpr Try CYSD

Met ASP Glu Arg His Lps

Bovine milk Lact.oferrin (B) 80,000 50 66 44 35 35 41 14 64 30 28 15 17 3 68 74 39 11 60

Human milk Lactoferrin

(GI

(B) 88,000

(M) 95,000

48 64 40 34 32

66 72 52 35 42 50

72 95 60 37 43 61

86,000

44

16 62 26 20 15 18 5 64 66 36 10 49

28 37 28 1 or 0 16 6 81 88 53 12

56

96 36 9 15 -

92 76 53 16 51

Human blood Serum transferrin

(P)

90,000 51

59 42 32 36 47

i:: 28 26 8 40 10 82 62 28 19 60

a B = Blanc, Bujnrd, and Mauron (1963) (preparation of Gordon); G = Gordon, Groves, and Basch (1963); M = Montreuil, Biserte, Mulet, Spik, and Leroy (1961); P = Parker and Bearn (1962).

specific. The proteins designated as serum transferrin (milk) have all been shown by immunoelectrophoretic methods to be similar to (if not identical with) serum transferrin. While lactoferrin has been isolated in sufficient quantity for precise chemical and physical studies, serum transferrin (milk) has not yet been isolated in adequate quantities or with sufficient purity for comparative studies of its chemical and physical properties with those of blood serum transferrin. (2) Bovine serum transferrin was first shown by Ashton (1958) and Hickman and Smithies (1958) to exist in a number of genetic variants. Similar observations were made by Smithies (1957) for human serum transferrin. It is apparent from the work of Groves (1965) that such polymorphism occurs in the case of serum transferrin prepared from the milk of a given cow, its electrophoretic pattern being identical with that of serum transferrin from the blood of the same animal (see Fig. VI-2lb).

204

H. A. MCKENZIE

A similar conclusion was reached from the genetic studies of Gahne et al. (1960). Starch gel electrophoresisrevealed three to five bands (A, B, C, D, E) in the serum transferrin patterns from blood of individual animals. Variation has been shown to be controlled by three autosomal alleles with no dominance (TfA,TfD,TfE). The allele TfA causes the appearance of bands A, B, and C; allele TfDof bands B, C, and D; and T f E of bands C, D, and E. ( 3 ) Groves el al. (1965) have shown that bovine lactoferrin exhibits genetic polymorphism. ( 4 ) Gordon el a.?. (1962) found that the iron-free form of lactoferrin can be isolated from bovine milk. It is a colorless protein, and resembles the iron-protein in chemical and physical properties. ( 5 ) The iron is dissociated from lactoferrin at ca. pH 2 compared with pH 4-6 for serum transferrin. (6) The amino acid compositions of the various proteins are similar, but there are striking differences in contents of tryptophan and methionine between human and bovine lactoferrin, and in amino acid composition between human lactoferrin and human serum transferrin. There is a considerable difference in histidine content between the lactoferrins and human serum transferrin. (7) The only precise physical study of the heterogeneity of lactoferrin is that of Szuchet-Derechin and Johnson (1966b). They studied sedimentation diagrams of one of their lactoferrin fractions. (Now that we know there are genetic variants of lactoferrin, it would be interesting to know whether this is a single variant.) The method of Fujita (1959) indicated that their preparation was “highly homogeneous.” They concluded the protein molecule was little hydrated.

4. Lactoperoxidase The green iron-binding enzyme of milk was first isolated by Theorell and Wkeson (1943) and afterward by Polis and Shmukler (1953). It was originally believed to be a degradation product of one of the red proteins and appeared to be heterogexieous. A single form of the enzyme was later isolated by Morrison and Hultquist (1963). Groves’ (1965) recent fractionation of bovine whey protein by chromatographic procedures (see subsection 3 above) indicates that this enzyme can be isolated readily from his fraction 1F. Recent preparations of the enzyme appear to consist of a single antigenic component distinct immunologically from lactoferrin (Allen and Morrison, 1963a,b). The enzyme has a molecular weight of ca. 82,000, a nitrogen content of 15.6 %, an iron content of 0.069 yo,and an isoionic point of 9.6. Its optimum activity appears to be near pH 6.8.

MILK PROTEINS

205

It is a heat-stable enzyme and could be a useful indicator for hightemperature heat treatments of milk. 6. Summary of Iron-Binding Proteins

There are two kinds of red-colored iron-binding protein in bovine and human milk whey: one, lactoferrin, is specific to milk; the other, serum transferrin, is similar to serum transferrin prepared from the blood of the same animal. This situation may not be the same in all animals (cf. Ezekiel, 1965). Further work on a variety of mammals is necessary. Since both types of red iron-binding protein exhibit genetic polymorphism, isolation of homozygous proteins is clearly called for; accurate chemical, physical, and immunological work will be necessary. The molecular weight of 90,000 needs close study: it will indeed be unique if this represents the minimum molecular weight of a single protein chain. The green iron-binding protein of milk, lactoperoxidase, is the most abundant of the enzymes in milk.

F. Lactollin During isolation of lactoferrin from mature bovine milk, Groves (1960) observed a crystalline protein associated with the red fraction in very small amounts. Groves et al. (1963) have reported the isolation and properties of this protein, which they termed lactollin. The method of isolation involved extraction of acid casein at pH 4.0, ammonium sulfate fractionation, and chromatography on DEAEcellulose columns, from which it was eluted immediately following the lactoferrin. The yield was ca. 0.5-1.5 mg/liter of mature milk. However, they were able to obtain about 4 times this yield from colostrum. At pH 9.5 single symmetrical peaks were observed on moving-boundary electrophoresis of lactollin, whereas at pH 7.0, 4.9, and 3.5 two or more peaks were observed and the patterns were nonenantiographic. In starch gel at pH 3.7, in the presence of 5 M urea, only a single band was obtained. The isoelectric point was found to be 7.1; the region of minimum solubility was near pH 8. Sedimentation velocity measurements gave a value of S Z O , ~= 3.21 S for a concentration of 10 gm/liter in pH 5.0 acetate buffer ( I 0.1). Some evidence of a timedependent polymerization was obtained. The diffusion coefficient was ca. 6.83 F, the calculated 5 0.734 ml/gm, giving an apparent molecular weight of 43,000 f 5000. Lactollin contains 16.4 % N, <0.1 % P, 0.14 % hexose and 0.15 % hexosamine. Its amino acid composition is unusual in that it contains no methionine, very little alanine and cystine, and a large amount of aromatic amino acid residues. The minimum molecular weight from the

206

H. A. MCKENZIE

amino acid composition is 10,900, One cannot help but wonder if the observed particle weight of 43,000at pH 5.0 is that of a tetramer. At present there is no known biological function for lactollin. I n view of its composition and its much greater preponderance in colostrum, it seems likely that it is not synthesized in the udder and that it may be related to the immune proteins.

G . Serum Albumin It was realized early that there is a protein in milk whey with properties similar to those of serum albumin. This albumin was crystallized by Polis et al. (1950) and Coulson and Stevens (1950) showed it to be similar serologically to blood serum albumin. Larson and Kendall (1957) found the secretion of serum albumin in bovine milk to be highest on the day of parturition. The level drops quickly and becomes about 1 % of the total protein in mature milk. Lunsford and Deutsch (1957) subjected human milk rennin whey to electrophoresis and separated out the slowest moving material. It was immunologically similar to human serum albumin. Johansson (1958) separated out a protein fraction from human milk by ammonium sulfate fractionation and calcium phosphate chromatography. This fraction had the same electrophoretic mobility as human serum albumin and similar immunological properties. Gugler et al. (1958) and I-lanson (1960) made an immunological identification of human serum albumin in colostrum and mature milk. Gugler and von Muralt (1959) showed that human colostrum contains about 2500 mg of human serum albumin per liter and that by the third to seventh day of lactation this falls to 200-300 mg/liter, i.e., about the same level as in bovine milk (ca. 330 mg/liter). Bell and McKenzie (1964, 1966b) identified serum albumin in ovine milk. Bell and Stormont (1965) found evidence of genetic variants of serum albumin in equine milk. There seems to be little doubt that the serum albumin in the milk of these species, at least, is identical to the serum albumin of the blood of the particular species. (The reader is referred for a detailed discussion of the properties of serum albumin to the comprehensive review of Putnam, 1965.) VII. SOMEENZYMES OF MILK

A . Introduction Milk contains a wide range of enzymes (see Section 11),some of which are secreted by the mammary gland whereas others are of bacterial origin. Some, such as the lipase and protease, act upon substrates present in the

MILK PROTEINS

207

milk, others need substrates foreign to milk. The most abundant enzyme in milk is lactoperoxidase (considered in the previous section, among the iron-binding proteins). Of the other enzymes present, alkaline phosphatase has been studied fairly extensively, since milk is a convenient source of this enzyme and its sensitivity to heat makes its activity a convenient index by which to judge the extent to which milk has been heated in pasteurization, etc. Milk xanthine oxidase has also been studied, since milk is a rich and easily accessible source of this enzyme, which plays an important part in the oxidation and transhydrogenations of pyridine nucleotides and is suspected to be a possible cause of deterioration in milk flavor on storage.

B. Acid Phosphatase Acid phosphatase (EC 3.1.3.2) occurs in small quantities in milk and is confined to the nonlipid phase. The concentration falls as lactation proceeds. This enzyme is particularly stable to heat, requiring some 30 minutes a t 80” for inactivation. However, it is inhibited strongly by light (Mullen, 1950). Bingham and Zittle (1963) purified milk acid phosphatase by adsorbing it from skim milk onto Amberlite resin, concentrating it further with acetone and then purifying it by chromatography on Amberlite resin and eluting with ammonium acetate. The preparation was very active on phosphates where the phosphate group is esterified with a phenolic hydroxyl group. The pH range of optimum activity was 4 . 6 4 . 8 . Pyrophosphate was attacked also. The preparation was twice as active in dephosphorylating casein as alkaline phosphatase. I t was activated by ascorbic acid, but not by thioglycolate or cysteine.

C . Alkaline Phosphalase The alkaline phosphatase concentration in milk is very variable: there can be some 40-fold variation in t,he content of milk samples taken from an individual cow at different milkings. The concentration of the enzyme seems to be in inverse relationship to the milk yield and does not appear to depend on the breed, feed, or fat content (Jenness and Patt,on, 1959). Morton (1953, 1954, 1955) studied the enzyme extensively and found it to be associated entirely with a particulate fraction of a lipoprotein nature. Electron microscopy studies indicated that these particles were <30-200 mp in diameter. Morton called the brown lipoprotein particles “milk microsonies” ; they were found to contain also xanthine oxidase, diaphorase, cytochrome c reductase, and a hemochromogen, possibly cytochrome c. Morton released the alkaline phosphatase from the lipoprotein particles into solution by utilizing his classical butanol treatment at 35”. The

208

H. A.

MCKENZIE

enzyme was found to have an optimum activity at pH 9.65. It hydrolyzed all true orthophosphate monoesters and also phosphocreatine but not the pyrophosphates. He found that magnesium(I1) and manganese(I1) activated it, but that cysteine inhibited it. Zittle and Bingham (1959) studied the splitting of phosphate from casein and phosphoserine by alkaline phosphatase. Their purified alkaline phosphatase dephosphorylated casein readily when the enzyme concentration was ca. 10 times that in milk; however, at 1/200 of this concentration the enByme acted on phosphoserine. Zittle and Bingham drew attention to the possibility of dephosphorylation of casein by the enzyme in milk and the effect of this on the phosphate content of the isolated casein. Andrews (1964) estimated the molecular weight of the enzyme, by Sephadex-gel filtration, as 148,000. No studies appear to have been made of the enzyme in dissociating solvents. When milk is exposed briefly to high temperatures there is loss of alkaline phosphatase activity. If the milk is then cooled and stored, the enzyme activity tends to reappear gradually. Reactivation does not usually occur if the milk has been heated to 63" for 30 minutes or to 72" for 15 seconds, Thus effective pasteurization usually destroys the alkaline phosphatase activity. Lyster and Aschaffenburg (1962) studied the reactivation of the enzyme. They found that the behavior of the isolated enzyme was different from that in the presence of milk. Magnesium(II), Sglycerophosphate and @-lactoglobulin were found to be necessary for reactivation. Serum albumin or casein was effective instead of $lactoglobulin, but a-lactalbumin was not satisfactory. Lyster and Aschaffenburg found an activator in the aqueous phase of milk, which was capable of enhancing reactivation and of replacing p-lactoglobulin in the role of activator for the purified enzyme. The activator was nondialyzable. They also found an inhibitor in the aqueous milk phase, and it was dialyzable. Richardson et al. (1964) have considered the problem of alkaline phosphatase reactivation. They suggest that calcium(I1) and magnesium(I1) are essential for reactivation and function as activators or coenzymes. It is suggested that these ions are removed from the enzyme during heat treatment as colloidal particles, and that in subsequent storage the ions gradually recombine with the apoenzyme.

D. Amylase Guy and Jenness (1958) found that a-amylase is present in bovine milk in the fraction precipitated from whey by ammonium sulfate a t 43 % saturation. I t was dissolved in an ethylene glycol-ethanol-water mixture, adsorbed onto rice starch and eluted with saturated calcium sulfate solution. The concentration of this enzyme in bovine milk is variable; it is

MILK PROTEINS

209

particularly high in the milk of cows suffering from mastitis. It requires calcium(I1) and chloride for activity and has optimum activity a t pH 7.4 and 34". It is inactivated at pH 6.4 in the temperature range 45-52". Guy and Jenness also found that some cow milk contains weak &amylase activity. Human milk is considerably richer in a-amylase than bovine milk (Wuthrich el al., 1964).

E. Lipases The lipases (EC 3.1.1.3) present in bovine milk are of considerable interest in milk processing and storage, as they are responsible for undesirable rancid flavors in milk, alter the surface and interfacial properties, and are important in the development of desirable flavors in cheese. They also occur in human milk, at several times their concentration in bovine milk (Heyndrickx, 1962). Naturally the milk lipases have received a good deal of attention, as can be seen in the recent comprehensive reviews by Chandan and Shahani (1964) and Jensen (1964). Since milk lipases have been obtained in a reasonably homogeneous state only quite recently, very little attention has been directed so far to their characterization. Two kinds of lipolysis have been distinguished in bovine milk : induced and spontaneous. The lipase of induced lipolysis is considered to be associated with the casein micelles. Certain treatments of the milk are necessary to effect adsorption of the enzyme to the substrate (milk fat) for the initiation of lipolysis. Homogenization, agitation, and foaming of warm milk have been found to be effective (Krukovsky, 1961). All mature cow milk is subject to induced lipolysis. On the other hand, spontaneous lipolysis occurs only in the milk from a small proportion of individual cows. In spontaneous lipolysis, the action of lipase on the milk fat globule membrane is initiated by merely cooling the freshly drawn milk (see Tarassuk and Frankel, 1957). Tarassuk et al. (1964) have shown that spontaneous lipolysis on cooling is a general phenomenon in human milk, in contrast to cow milk. Thus human milk intended for cold storage will become rancid unless it is pasteurized. The inactivation of lipases in milk on aging and heating has been attributed to the destruction of SH groups essential for lipase activity. Although the Iipase causing induced lipolysis in cow milk requires some treatment of the milk in order to hydrolyze the fat globules, no treatment is necessary in order to attack simple esters added to the milk. Its activity is affected by heat, light, irradiation, and constituents of the milk (Jenness and Patton, 1959). Hetrick and Tracy (1948) claim that, while pasteurization inactivates this enzyme, it becomes reactivated on storage. Dorner and Widmer (1932) first found that this enzyme was associated with

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casein. Yaguchi et al. (1964) fractionated dialyzed skim milk on DEAEcellulose columns and found that this lipase activity is associated with the K-casein fraction. The lipase activity in various fractions was roughly proportional to the sialic acid content of the fraction. They concluded that the major milk lipase must be an SH-containing enzyme associated with K-casein, and suggested that rennin splits K-casein into a lipase-active para-K-casein and a lipase-inactive glycomacropeptide. They believe that para-K-casein has one easily oxidizable SH group, essential for the lipase activity. Chandan and Shahani (1963a,b; also Chandan et al., 1963) prepared a lipase from milk clarifier slime and fractionated it by chromatography. , ~ Their preparation gave a single peak in the ultracentrifuge with an S O ~ O = 1.14 S and had a molecular weight of ca. 7000 at pH 8.5 (0.1M Tris-maleate) and 20". It was active in the p€I range 5.2-9.8 with an optimum activity at pH 9.2 and 37". Shahani and Chandan (1965) found K-casein, milk 7-globulins, and blood serum albumin stimulated it, whereas a-lactalbumin, &lactoglobulin, whole acid casein, and a-,as-,b-, and ?-caseins inhibited it. Nelson and Jezeki (1955) found no direct correlation between the lipase activity in clarifier slime and in whole raw milk. This observation, taken along with the low molecular weight of 7000, caused Yaguchi et al. (1964) to doubt that the lipase isolated by Chandan and Shahani is the major lipase of milk. Gaffney and Harper (1965) investigated separator slime; they found that it consisted of a large amount of somatic cells and that these cells were rich in an endolipase found to be different from the lipase of skim milk by polyacrylamide gel electrophoretic and substrate specificity studies. Downey and Andrews (19654 found that tributyrinase activity in bovine skim milk is associated mainly with the casein micelles, but that supernatant solutions containing about 70 yo of the total tributyrinase activity can be obtained by adding sodium chloride (final concentration 0.75 M ) to skim milk and centrifuging at 80,000 g for 1 hour. Partial separation was achieved by gel filtration on Sephadex G-200 columns. Most of the lipase activity seemed to be due to up to five enzymes, with low substrate specificities, although there were some differences in relative activity toward tributyrin, triolein, milk fat, and triacetin. One lipase was estimated tentatively as having a molecular weight of 4000 and possibly similar to that of Chandan arid Shahani, while the others were estimated to have molecular weights of 50,000-130,000. It is possible that there is only one enzyme, that it has a low molecular weight, and that it exists in a free form or is associated with casein or other proteins. Unfortunately experiment,s, along the lines suggested for such systems in Section IV, were not carried out to gain further light on this.

21 1

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Downey and Andrews (196513) demonstrated the reversible association of lipases with casein micelles. The enzymes were eluted during gel filtration in 0.1 M sodium chloride almost entirely with the casein. However, in 0.2 M sodium chloride the enzymes were eluted after the casein fractions and in positions corresponding to those of the free enzymes. The ability to associate with casein seems to be a property of lipases since, when pancreatic lipase was mixed with a milk lipase preparation in 0.1 M NaCI, it was eluted in gel filtration with the casein. However, when it was mixed into a preparation from which the casein had been removed, it was eluted in its normal position. The possibility that the casein micellar structure is necessary is suggested by the failure of pancreatic lipase to associate with purified a-,p- or K-casein.

F. Lysozyme Lysozyme (EC 3.2.1.17) has been detected in milk from a very large number of species. Much of the earlier work resulted in conflicting results. More recently Jollks and Jolles (1961) separated the enzyme from human milk by chromatography on Amberlite CG-50 at pH 6.5, followed by extensive fractionation on CM-cellulose. The purified enzyme had properties similar to those of lysozyme, previously isolated from sources other than milk. It was very stable a t low pH, being unaffected by 3 minutes a t 100" and pH 4.5. As the pH was raised it became unstable, e.g., 30 % activity was lost after 1 minute at 100" and p H 7.5. Preliminary studies indicated a molecular weight of the order of 15,000 and an amino acid composition similar to, but not identical with, that of egg white Iysozyme. Shahani et al. (1962) found the lysozyme content of cow milk to be highest among the 4-8-year-old cow group. There was variation in the lysozyme content with breed, but not with stage of lactation. Parry et al. (1964) modified the method of isolation of Jollks and Jollks. Chandan et al. (1965) compared the lysozyme content of human milk with that of bovine milk. The former was some 3000-fold richer in lysozyme: 390 mg/liter vs. 130 pg/liter. The human lysozyme was much more heatsensitive. The bovine milk lysozyme had S O Z O , ~= 2.0 S at p H 5.4 (0.15 M KC14.02 M NaOAc) and 25O, whereas the human enzyme had = 1.35 S. The isoelectric point of the bovine milk enzyme was pH 9.5 in contrast to pH 11.0 for the human milk enzyme. The p H values for optimum activity were 7.9 and 6.35, respectively. sOZO,,,.

G. Ribonuclease Bingham and Zittle (1964) found bovine milk to be a good source of ribonuclease (EC 2.7.7.16), although its concentration varies widely during the course of lactation. Two ribonucleases were found to be present, the

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major one being apparently identical to bovine pancreatic ribonuclease A. This conclusion was supported by electrophoretic, chromatographic, amino acid composition, and imniunological studies. The minor component, ribonuclease B, had the same amino acid coniposition as ribonuclease A, but contained carbohydrate in addition. Bingham and Zittle suggest that the milk ribonuclease is formed in the pancreas and transported to the mammary gland by the blood. Although bovine milk is rich in ribonuclease, there is only a trace in human, ovine, caprid, and porcine milk. Bovine milk ribonuclease is heat-stable a t pH 3.5, being able to stand 20 minutes a t go", but a t pH 7.0 all the activity is lost under these conditions. Zittle (1964) considers that the presence of ribonuclease in milk must affect the stability of the cream emulsion.

H . Xanthine Oxidase Morton (1954) found xanthine oxidase with the alkaline phosphatase in the milk microsomes attached to the fat globules. The enzyme can be released into the plasma by various treatmenk arid the distribution in a given sample of milk depends on its previous history. The distribution was also studied by Pasquier (1960). Less xanthine oxidase was found in human milk than in bovine milk. Pereira et al. (1962) found the enzyme primarily in the lipid of raw milk, but also detected it in skim milk and rennin whey. Gilbert and Bergel (1964) found that the addition of cysteine helped to release the enzyme from the fat globule. The enzyme is moderately stable to heat, but 25 % of its activity in milk is lost after 18 seconds a t 76" (Zittle, 1964). Milk xanthine oxidase contains protein, molybdenum, iron, and flavin (flavin :Fe :Mo = 2 :1:8) according to Avis et al. (1955). The particle weight of milk xanthine oxidase has been estimated as 275,000 (Andrews el d.,1964), but no attempt appears to have been made to determine it in dissociat,ing solvents. OF M r L K VIII. IMMUNOGLOBULINS

A . Introduction Ehrlich realized in 1892 that immune bodies passed from the cow via the colostrum to its offspring, giving it passive immunity. It was not until the excellent work of Smith (1946, 1948) that a systematic attempt was made to isolate and study the proteins in milk associated with immunity. He concluded that colostrum serves the special function of enhancing the resistance of the newborn to infectious disease. He showed that the

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distribution of proteins in colostrum is quite different from that in mature milk. The total protein content of colost
B. Immunoglobulin Types an Milk In human colostrum there are a number of proteins in common with blood serum. These are summarized in Table VIII-1. The immunoglobulins present in colostrum consist mainly of the yA(ylA: BzA) and yM(ylM; &M) types and a smaller amount of a yG(yss; 75,) globulin. In mature human milk the main type is yA(ylA; P2A) (Hanson, 1960; von Muralt et al., 1964). Identification has not been complete. Gugler and von Muralt (1969) and Hanson (1961) detected six different types in the immune globulin region by immunoelectrophoretic methods. Four of these appear to re-

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semble blood serum proteins; two are specific to milk. Further identification of the latter must await their isolation in an adequately pure condition. Lubin et al. (1964) fractionated the immunoglobulins of human milk and isolated two fractions containing protein specific to milk in addition to several common to blood serum [yA(ylA; ,&A), yM(ylM; &M), yG(yss; 7Sy)I. The two milk-specific proteins had antigenic determinants related to, but different from, serum yG globulin. TABLE VIII-1 Serum Proteins Present in Human Colostrim and Milka Protein

Colostrum

Human milk

prealbumin albumin al-acid glycoprotein (orosomucoid) ( ~ ~ - 3 .glycoprotein 5s az-macroglobulin az-lipoprotein ceruloplasmin (az-globulinI haptoglobulin (az-globulin) transferrin fibrinogen pl-lipoprotein p,a-globulin rA(ylA; &A)-globulin rM(ylM; pzM)-globulin rG(yss; 7Sy)-globulin (rapidly migrating) yG(yss; 7Sy)-globulin (slowly migrating) Modified from von Muralt et al. (1964); (+) = trace, ((+)) = smallest trace only detected by enrichment procedure.

Using chromatographic procedures, Hanson and Johansson (1962) and Montreuil et al. (1960a) isolated yA(ylA; PZA) globulin from human milk. Montreuil and co-workers considered this protein and the blood serum protein to be identical. However, the Swedes reached the conclusion that, although the proteins are very similar antigenically, there is no strong proof that they are identical. They also found isoagglutinins (the blood group antibodies) associated with the milk yM(ylM; &M) globulin (as is the case in blood serum). I n man, immunoglobulins such as those associated with the polio virus antibody may occur in the milk when the antibody is very weak in the blood serum. Sabin and Fieldstiel (1962) found no correlation between the concentration of the antibody in colostrum and in blood serum (cf. Warren et al., 1964).

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C . Importance of Immunoglobulins in Milk In the rabbit, guinea pig and man, transfer of antibodies takes place across the placenta, so that antibody content of the mother’s milk has not been considered generally as an important source of immunity for t,he newborn child. However, in the horse, pig and ruminants, no placental transfer occurs and the colostrum is the only source of immunity for the new animal. This source is available only for a short time because the level of immunoglobulins in the milk drops very quickly and after 36 hours the gut of the animal seems to be unable to absorb the large particle weight antibodies. Human milk has been found to possess marked antiviral properties; it inhibits growth of mumps, influenza vaccinia, herpes simplex, and several encephalitis viruses. These “milk factors” appear to be mucopolysaccharides. However, antibodies to poliomyelitis and Escherichiu coli have been found in the protein fraction of milk. Lubin el al. (1964) found the yA(ylA;P2A) globulin to be associated with the antibody activity against type I poliovirus, and the rA(y1A; PA) and rM(y,M; &M) types to be associated with the bacterial hemagglutinating antibody of E . coli. The antipoliomyelitis factor occurs in the milk of mothers with circulating serum antibodies to poliomyelitis. Passive transfer occurs through the milk to the newborn infant. Sabin and Fieldstiel (1962) and Warren et al. (1964) consider that the presence of these antibodies in the gut of the newborn (feeding on its own mother’s milk) prevents establishment of the poliovirus. In such cases successful vaccination with attenuated virus cannot be carried out until the child is ca. 6 weeks old (by this time the antibody level of the mother’s milk has fallen and protection decreases). It is thought that protection against other enterovirus infections may occur and explain at least in part the reduced morbidity of breast-fed babies. Hodes et al. (1964) isolated the poliomyelitis antibody of human colostrum. They claim that it is different physically and chemically from that of blood serum. Campbell and Petersen (1963) have claimed for many years that cow milk rich in immunoglobulins can cure adult human subjects of rheumatoid arthritis and hay fever. Their theory depends on the assumptions that the adult gut can absorb immunoglobulins and that it is possible for cows to produce milk rich in immunoglobulins following injection of antigen into their udders. Lascelles (1963) has made a thorough investigation of these claims and has produced compelling arguments against them. The study of the immunoglobulins of milk is a fascinating one, hardly

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opened up. Thorough investigation of these proteins using modern methods should yield rich dividends.

IX. MILKPROTEINS AND ALLERGENICITY REACTIONS A . Introduction A t the beginning of this review it was pointed out that, because some mothers do not breast feed their babies, human milk substitutes such as cow milk are used, and these substitutes give rise to numerous problems. Unfavorable reactions to the ingestion of bovine milk by infants have long been known. Hippocrates noted a case of urticaria attributed to bovine milk. In more recent times such reactions have been referred to as milk allergenicity reactions. Milk may be the incitant but in many cases the reaction may not be an allergic one, since the body may be only displaying intolerance to lipids, salts, sugars, or poor preparation of the milk. It is difficult to assess the frequency of true allergic reaction to milk, since clinical diagnosis is dependent on cure of the condition when milk is removed from the diet. Skin tests are unsatisfactory, since positive reactions are often obtained where no clinical history of milk serisitivity exists. Passive cutaneous transfer, which reveals nonneutralizing or non-precipitating antibodies, and anaphylaxis in sensitized guinea pigs, have been widely used. However, intensity of reaction is influenced by a variety of conditions that cannot be rigidly controlled. No satisfactory Iaboratory tests are presently available to aid in diagnosis of allergenicity or in,assessment of power of allergenicity of milk proteins (Sheehan and Glaser, 1963; Collins-Williams and Salama, 1965). This difficulty of diagnosis has resulted in varied opinions as to the frequency of milk allergenicity. Some consider it to be very common and of great importance, settiiig the life pattern of the health of the infant. Others consider it to be overemphasized and to be important only in 0.3 yo of infants. Hanson (1960) and von Muralt et al. (1964), using immunoelectrophoretic techniques, found evidence for at least 10-15 antigens in human and bovine milk that are related to human serum, and some 7-8 antigens specific to milk. Molecular size can influence antigenicity, because if a small molecule is absorbed into the gut it may be eliminated quite quickly by glomerular filtration in the kidney. Likewise various physiological conditions (e.g., presence of other foods in the stomach) can affect the severity of an allergic reaction.

B. Milk Protein Antibodies an Serum Circulating antibodies (precipitating, agglutinating, complement-fixing, and nonprecipitating) to bovine milk proteins have been detected in the

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sera of normal and diseased human subjects. Antibodies to human milk have not been detected in the sera of infants fed on cow milk (Gold and Godek, 1961; Gunther et aE., 1962). No antibodies to bovine milk have been found in the sera of breast-fed infants (Gunther et aZ., 1962). The significance of the antibodies to milk proteins in human sera is doubtful, as great variation has been obtained in the assessment of their occurrence, partly due to variation in sensitivity of the various techniques used. Some workers (Gunther et al., 1960) have found the antibodies in the sera of 98 % of the children studied, and others (Holland et al., 1962) in only 5 %. Saperstein et al. (1963) found no relationship between serum antibody level and susceptibility to milk allergy, using the Boyden tanned cell hemagglutination and the Crowle double diffusion in gel tests. However, the PCA (passive cutaneous anaphylaxis) test for nonprecipitating antibodies (rG) gave a correlation with the incidence of milk allergy. Holland et al. (1962) and Peterson and Good (1963), using double diffusion in agar and immunoelectrophoretic techniques, found that the presence of precipitins was correlated with certain pathological symptoms. Heiner et al. (1962), using a modified Crowle technique, showed an elevated level of antibodies in the sera of children suffering from celiac disease. The effect in the latter two cases could be secondary to the disease, increased absorption of milk protein by the gut occurring due to the disease. Possibly a true assessment of susceptibilit,y to milk allergy is not obtained by measuring circulating antibodies, because these give only a measure of a secondary manifestation in vitro of the primary reaction between antigen and antibody. In an endeavor to measure the primary interaction, Rothberg and Farr (1965) examined the precipitation of Plabeled bovine serum albumin and bovine a-lactalbumin antibody complexes by the sera of 900 children and adults. About 75 % of the children were found to have the antibodies. Anti-bovine serum albumin was twice as frequent as anti-bovine a-lactalbumin. Both were 3 times as frequent in the under-16 age group as in the 16-40 age group. Other investigations of antibodies in human serum have also given interesting information. In a group of 108 newborn babies, Gunther et al. (1962) found a marked increase in the number of agglutinating antibodies to bovine milk in the sera when the infants were fed cow milk. Antibodies to casein, a-lactalbumin, and p-lactoglobulin were identified. Relatively large amounts of antibodies were formed in the first few days, when antibodies to virus and bacterial diseases are not usually formed. Gunther and co-workers suggest that the normal immune response may be overwhelmed by the antigens of the bovine milk. I t is hoped that this work will be carried further, since breast-fed babies have a greater resistance to disease at this initial period of life and the

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reason is not clearly understood. The suggestion was discussed (Section VIII) that protective antibodies in the mother’s milk may prevent multiplication of enteric bacteria and viruses in the gut, and so give the newborn child passive protection without need for the antibodies to be absorbed into the blood stream. It would be interesting to know if the substitution of cow milk for mother’s milk harms the child’s protective mechanisms. Some authorities consider that the so-called “cot deaths” (Great Britain) or “crib deaths” (United States) are due to hypersensitivity to milk, These deaths were attributed by Barrett in 1954 to inhalation of regurgitated food into the lungs, setting up an inflammatory reaction, possibly in the nature of a hypersensitivity reaction. Parish et al. (1960a) have shown that sensitized guinea pigs, following light anesthesia, die peacefully and exhibit similar lung pathology to that found in crib deaths if milk is added to t~helungs. Parish el al. (1960b) studied a numker of cases of crib death. They found that the sera of these “crib death” infants had a higher titer of agglutinating antibodies to bovine milk and a higher level of incomplete antibodies, and gave more positive PCA tests (now known to be correlated with the presence of r G globulins), than the sera of a group of normal healthy infants (Parish et al., 1964). Coe and Peterson (1963) and Gold and Godek (1961) obtained pathological and serological findings contrary to those of Parish and his colleagues. Gold and Adelson (1964), in a further extension of this work, drew attention to the presence among Parish’s normal infants of 32 % who had never been bottle fed but were breast fed for the first 6 weeks of their lives, before transferring to cow milk. Gold and Adelson therefore investigated the level of bovine milk antibodies in children fed human milk for their first 6 weeks, then cow milk. These infants displayed a lower level of bovine milk antibodies than those fed cow milk from birth. In the light of these contradictory findings, it seems that further careful work is necessary to resolve the issue.

C. Allergens in Milk There has been considerable effort directed toward finding the principal antigens in bovine milk causing the allergenicity reaction in human subjects. Attempts have also been made to alter the allergen by heating the milk or by other simple treatments prior to its use by babies. As far back as 1905, pediatricians in Paris and Vienna were blaming the whey proteins for causing milk allergies. Ratner (1935) considered that milk lost its antigenic properties on heating, and that (‘lactalbumin” was the strongest allergen in mature milk. It is now obvious that the purity of protein preparations, over a greater part of the period in which the late Dr. Ratner worked, was insufficient, so that little reliance can be placed on findings

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obtained with them (e.g., bovine serum albumin is a powerful antigen and many of the preparations of other milk proteins contained it as an impurity). Furthermore, whether tests are carried out by oral challenge or by intravenous injection can greatly influence the results. Comparisons have been made of the allergic strength of many of the milk proteins-e.g., casein, a-lactalbumin, and p-lactoglobulin (Ratner et al., 1958; Parish et al., 196Ob; Crawford and Grogan, 1961a; Cole and Dees, 1963). None of the proteins was shown to be a dominant allergen. Most of these comparisons are subject to the above criticisms. Goldman el al. (1963) carried out a marathon study and found little differences in the allergenicity of these proteins. L. W. Hill (1964) has made criticisms of this work similar to those made by the reviewer above. The effect of heat on the allergenic properties of milk has been studied by a number of investigators. Among the more reliable investigations are those of Roulet et al. (1961), Hanson and hlansson (1961), and Saperstein and Anderson (1962). The first two groups studied the effect of heat on bovine milk and bovine milk products by immunoelectrophoretic techniques. Both groups found the y-globulins to be the most sensitive to heat treatments, whereas the major whey proteins, p-lactoglobulin and a-lactalbumin, were relatively heat-insensitive. Both these groups of workers and Saperstein and Anderson found that various nonallergenic” baby formulas contained milk protein allergens. Goat milk has also been recommended as a l‘nonallergic” milk, for it was originally thought that goat’s milk did not contain the suspected whey protein allergen. However, work of Saperstein (1960), Crawford and Grogan (1961b), and Hanson and Andersen (1962) has shown immunologically that caprid and bovine whey proteins are closely related. Bell and McKenzie (1964) have found that there is no p-lactoglobulin in human milk (see also Johansson, 1958). This work indicates that any role of specific whey proteins as dominant allergens needs careful reassessment. (‘

x.FUTURERESEARCH Progress in our detailed knowledge of individual milk proteins in the last decade has been truly dramatic. Even fifteeI! years ago, if a worker in the field had hazarded the guess that within two decades we would have detailed knowledge of the amino acid composition and sequence and conformation of a number of milk proteins, his fellow workers would probably have suggested that he see his psychiatrist. Nevertheless such a goal is in sight. New techniques have enabled the detection of genetic variants of a protein where there are only one or two amino acid residue differences in the chain. Fortunately, the substitutions have been such that the charge on the protein has been affected sufficiently to enable resolution

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to be obtained with available techniques. More subtle differences in possible future variants may demand more sensitive techniques. Eternal vigilance for better physical, chemical, and biological methods will be necessary if progress is to continue. Some of the whey proteins are amenable to detailed X-ray crystal structure analysis. This will be achieved only if painstaking search is made for appropriate heavy metal derivatives. Fundamental studies of the preparation of such derivatives are sorely needed. Detailed studies by a variety of techniques, ranging from simple solubility studies to circular dichroism, will give an insight into the mechanism of the many subtle changes in conformation that milk proteins are able to assume. Their association-dissociation and aggregation reactions should be studied more carefully in the light of existing theories for such reactions. At the same time, it is hoped that some of the unexplained aspects of these reactions will lead theoretical workers to refine existing treatments and to develop new theories. The rewards will be rich, since the theories will not only be applicable to milk proteins but will have repercussions in protein chemistry generally. We have reached a clearer understanding of the mode of action of rennin on casein. It is well to recall that most of the advances have been made by examination of derivatives of K-casein preparations. Many but by no means all of the conclusions reached will be relevant to the clotting reaction in the complex environment of milk itself. There has been controversy in the past on the role of individual milk proteins in allergenicity and immune reactions. Much of this controversy has arisen from the low purity of the protein samples used. Even a trace of another protein can frequently ruin the validity of such experiments. The problems in this area are formidable, but unless proteins of the highest possible purity are available there will be no chance of success in the understanding of the reactions involved. In our greater knowledge of the chemistry of individual proteins isolated from milk, we all run the real danger of losing sight of their ultimate natural environment: milk itself. Only when we can understand the many reactions of whole milk will we be able to say that we understand the proteins.

ACKNOWLEDGMENTS The author is grateful to Professor W. Kausmann for making it possible for him to spend a term a t the Frick Chemical Laboratory, Princeton University, during which the preparation of this review was helped considerably. Assistance was received from a National Science Foundation Grant to Professor Kausmann. Special thanks are due to Dr. Margaret R. McKenzie for her help in many ways, particularly in assessment of the literature in Sections VIII and IX. Thanks are due to colleagues and students for assistance, helpful discussion, and

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permission to quote results prior to publication, and to the Australian Dairy Board for generously supporting the author’s work. Grateful acknowledgment is due to the authors and publishers who have permitted reproduction of diagrams from their papers and books.

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ADDITIONAL READING Since this article was prepared a number of important publications have appeared. The reader’s attention is drawn to the following articles: Rose, R., and Colviri, J. R. (1966). “Size and structure of casein micelles,” J . Dairy Sci.49, 351 and 1091. Berridge, N. J., and Suett, D. L. (1966). “Partition of casein between polymer phases,” J . Dairy Res. 33, 277. Schmidt, D. G., Both, P., and de Koning, P. J. (1066). “Fractionation and properties of K-casein variants,” J . Dairy Sci. 49, 776. de Koning, P. J., van Rooijen, P. J., arid Kok, A. (1966). “Location of amino acid differences in K-caseins A and B,’.’ Riochem. Biophys. Res. Commun. 24, 616. Peterson, R. F., Nauman, L. W., and Hamilt>on,D. F. (1966). “Amino acid composition of six distinct type of @-casein,” J . Dairy Sci. 49, 601.