NOMENCLATURE OF THE MILK--FIRST
PROTEINS REVISION
OF BOVINE
R e p o r t of the C o m m i t t e e on M i l k P r o t e i n N o m e n c l a t u r e , Classification, a n d M e t h o d o l o g y of the M a n u f a c t u r i n g S e c t i o n of A . D . S . A . f o r 1958-59 J. R. BRUNNER ~ (Chairman), C. A. ERNSTROM, ~ R. A. HOLLIS, 3 B. L. LA]~SON R. MeL. WHITNEY, ~ A:,-D C. A. ZITTLE a SUMMARY The a-casein component of the casein system of milk, which appears as a single, leading eleetrophoretie peak at p H 8.6, is heterogeneous and should be referred to as the a-casein fraction. The components comprising this fraction vary in distribution, dependent upon the experimental conditions. Alpha-casein has been separated into calciumsensitive and ca h./mn-insensitive fractions which have been designated by various symbols. These fractions usually are in the form of an ~ (Ca sensitive)--~ (Ca insensitive) complex in equilibrimn with its components. I n view of the complexity of the a-casein fraction, this Committee feels that no recommendations on nomenclature should be made at this time. Cherbuliez's 8-casein, Hammersten's proteose, and the 2% and 12/% TCA-soluhle peptides of Alais and Nitsehmann, materials apparently derived from a-casein by various procedures, are discussed in this report. /?-laetoglobulin, obtained from mixed milk, is composed of at least two forms of ~-lactoglobulin which are genetically determined and referred to as B-laetoglobulins A and B, discernible by p a p e r electrophoresis in veronal buffer at p H 8.6, where Type A constitutes the leading component. Further, Type A associates in acetate buffer between p H 3.7 and 5.2 and is essentially monomerie at p H values alkaline to its isoelectric point, whereas Type B exists in its monolneric form under similar conditions° These characteristics explain in part the electrophoretic and ultracentrifugal heterogeneity of normal (mixed) ¢-Iaetoglobulins A q- B and/~-lactoglohulin A.
A p r e v i o u s C o m n i i t t e e r e p o r t , J e n n e s s et al. (122), p u b l i s h e d in 1956, cont r i b u t e d m a t e r i a l l y to the c l a r i f i c a t i o n of a r a p i d l y e x p a n d i n g ' s y s t e m of nomenc l a t u r e for the p r o t e i n s of bovine s k i m m i l k . The e l e e t r o p h o r e t i c a l l y d i s c e r n i b l e p r o t e i n c o m p o n e n t s were c a t e g o r i z e d in t e r m s of t h e i r classical or m o r e traditio~lal n o m e n c l a t u r e a n d in r e l a t i o n s h i p to the c o n t e m p o r a r y n o m e n c l a t u r e . A l t h o u g h e l e e t r o p h o r e t i e r e s o l u t i o n of the p r o t e i n compo'nents in f r e e - b o u n d a r y electrop h o r e s i s was selected as a c o n v e n i e n t c r i t e r i o n f o r classification, the p o s s i b i l i t y was r e c o g n i z e d t h a t m a n y of these so-called i n d i v i d u a l p r o t e i n s were a c t u a l l y c o m p l e x e s or h e t e r o g e n e o u s m i x t u r e s of p r o t e i n s possessing s i m i l a r e l e c t r o p h o r e t i e c h a r a c t e r i s t i c s at p H 8.6. :Received for publication January 29, 1960. ~Department of ])airy, Michigan State University, East Lansing. Department of ])airy Food Industries, University of Wisconsin, Madison. 3 Research and Development ])iv., National Dairy Products Corporation, Oakdale, I, ong Isl,qnd. ~Department of Dairy Science, University of Illinois, Urbana. '~Department of Food Technology, University of Illinois, Urbana. Eastern Utilization Research Branch, VSDA, Philadelphia, Pa. 901
902
J. ~ . B R U N N E R
ET AL
Recent investigations have demonstrated the heterogeneity of the a-casein and fl-laetoglobuliln fractions. New, and at times confusing, terminology has been introduced by investigators to identify the proteins they have reported. The present Committee, in an attempt to cope with the problem of expanding terminology, recognized that: Within reasonable limits, the prerogative of the investigator to assign an appropriate nomenclature to the proteins he has isolated a n d : o r characterized should be preserved, provided that he shows conelusive evidence that his protein differs in fact, not purity, from any protein previously isolated and characterized; and the Committee's principal function was the resolution of newly introduced terminology in terms of contemporary nomenclature rather than recommending prematurely a rigid nomenclature system. Conceivably, newly reported protein fractions of similar characteristics could be classified tentatively pending: (a) the presentation of additional confirmatory evidence in support of the homogeneity of the protein, and (b) the development of a sound nomenclature system for milk pro'teins. A n awareness of the progress being made in this field would indicate that a stabilized nomenclature system must await a more complete elucidation of the milk protein system. Based upon the foregoing considerations, this Committee has co'nsidered the terminology introduced by investigators to designate the components of the ~-casein fraction and fl-laetoglobulin and has presented a compilation of this terminology in terms of contemporary usage. Also, certain additions and revisions have been made to the table of characteristics published in the 1956 report. In so doing, the Committee realized that f u r t h e r development will necessitate additional revisions, until a more complete elucidation of the milk protein system nmkes possible the establishment of a nomenclature and classification system which will facilitate research in the field of milk proteins. Caseim The 1956 report recognized the individuality of the components a-, fl-, and 7-casein in the protein fraction precipitated by acidifying raw skimmilk to p H 4.6. A fourth component, g-casein, isolated by Cherbuliez and his coworkers (7, 8, 9) attd characterized by its solubility in 10% TCA, was considered as a possible entity. The similarity between ~-casein and Hammersten's proteose, found as a product of the reaction of rennet on casein, was postulated (10). Alais (1) showed similarities between IIammersten's proteose, Cherbuliez's ~casein, and a 2% TCA-soluble fraction obtained by the action of crystalline rennin on casein. The bulk of the 2% TCA-soluble fractio'n stems from the ~-easein fraction (2) and more specifically front the calcium-insensitive fraction (K-casein) (57, 59) by the p r i m a r y action of rennin. The fact that Cherbuliez and Baudet (8) did not isolate 8-casein from paracasein suggests f u r t h e r that ~-casein and the rennin-liberated proteins may have similar origins. F u r t h e r investigations are required to establish the classification of a-casein. Within recent years several papers have been published elucidating the a-casein fraction. Waugh and yon Hippel (61) were the first to show that a-casein could be separated into calcium-sensitive [~-casein (59)] and calciuminsensitive [crude-K-casein] fractions, based upon its dissociation and differential solubility in the presence o'f calcium ions. The fractions of casein not classified
NOMENCLATURE
OF P R O T E I N S
903
as as am-, crude-K-, or fi-easein were designated m fraction (60). Long t~t aI. (30), working along a similar approach, were able to isolate a slow sedimenting fraction from Wangh's seeond cycle, crude-K-casein, characterized by its high phosphorus content (1.1%,), which they called X-casein. They suggested that the calciuminsensitive fraction of Waugh possessed properties similar to the ~-casein fraction isolated by Linderstr~m-Lang (29). The calcium-sensitive fraction of the a-casein fraction was called a~-(30) and, in a previoals study, ap-easein (61). Wake (57) has referred to the calcium-sensitive fraction as a-casein. MeMeekin et al. (33) reported the isolation of a fraetion from a-casein possessing minimum solubility at p H 5.8-6.0, but soluble at p H 4.7 as well as p H 4.0 (32), which they called a2-casein. This fraction was obtained from acid casein in yields approximating 1%, was low in phosphorus content (0.1%), not precipitated by calcium ions nor elotted by rennet, but was split by rennet at p H 7.3 into soluble and insoluble fractions. The remaining and larger fraction was called ~l-casein. Electrophoretic mobilities for the al- and a.~-casein fractions in veronal buffer at p i t 8.4, P/2 = 0.1 were - 6 . 7 and -5.0, respectively. MeMeekin (31, 32) has Since referred to .the a2-easein fraction as a=-casein. Characteristics of =~-easein suggest its similarity to I~inderstrCm-Lang's Z-casein and Waugh's K-casein. F u r t h e r , McMeekin (33, 34) referred to the calciumsensitive fraction of a-casein as a~-casein. More recently, t I i p p st al. (19) have reported the isolation of as-casein, characterized by a single ultraeentrifugal compm~ent at p H 7.1 (S._,o= 23 in phosphate buffer) and by its resemblance to K-casein in physical properties. Cherbuliez and Baudet (7) fraetionated a-casein into two fractions by warming it to 40 ° C. in 5% (NtI4),,SO, solution. The soluble (at) and insoluble (an) fractions were similar in composition and can not be considered as different protein entities. Nitsehmann and Lehmann (37) showed that rennin, when added to so'dium a-easeinate, induced an electrophoretically discernible split in t h e alpha component; the two peaks were called as and a,_, in descending order of mobility. Payens (40), studying the action of rennin on Waugh's first- and second-cycle casein fractions, concluded that Nitsehmann's ae--easein was quite similar, if not identical, to W a u g h ' s kappa-rich fraction. The split in the eleetrophoretie peak of the a-casein fraction, which has been noted by many investigators, indicates the presence of more than one component and presents data sometimes difficult to interpret, since any one or a conlbination of facto'rs, i.e., individual differences among cows, variations in ionic strength, freezing' or dehydration of the casein preparation, and prolonged storage of casein solutions, could contribute to the occurrence of such an observation (12, 58). Obviously, the a-casein fraction consists of a highly integrated system composed of ealcium~sensitive and calcium-insensitive protein components. Actually, a precise interpretation of the data reported in support of the various casein fractions is most difficult because of the tendency of casein components to form aggregates under all except very drastic conditions (20, 36). For example, it is difficult to' know whether a fraction with given properties is a pure component,
904
J . R. B I ~ U N N E R
ET AL
a mixture of components, or a fraction reflecting the specific conditions used in its isolation. Aggregation makes moleculer weight determinations especially troublesome and uncertain. In view of these advancements in the knowledge of the casein system, the classical term calcium para-easeinate should be redefined t o ' i n d i c a t e the specificity of the action of rennin (14, 57, 59, 50). Waugh et al. (59, 60) have proposed a new terminology for the action of rennin on the a-casein fraction: para-K-casein for the p r i m a r y reaction product of rennin on K-casein and a~para-K-easein for the clots which are formed. The role of other a-casein components and of fl- a n d / o r 7-casein in this transformatiou has not been elucidated. No recommendation relative to the nomenclature of the a-casein complex is suggested at the present time. As reviewed above and in Table 2, the a-casein complex is currently the subject o'f a great deal of research. I t appears desirable to withhold a recommended nomenclature for the a-casein complex until the components and physical/chemical equilibria involved are more precisely and completely understood. fl-lactoglobulin. Pedersen's (41) electrophoretic and ultraccntrifugal studies with Palmer's fl-lactoglobulin led him to believe that the protein was homogeneous. Li (28), employing more sensitive electrophoretic techniques, observed that fl-lactoglobulin showed a single eleetrophoretie peak in acetate buffer at p H 5.3 and 5.6, but showed three components when observed in buffer at p H 4.8 and 6.5. In both cases, the fastest-moving boundary constituted the major portion of the pattern and had an isoeleetric point of 5.1. Polls et al. (43) found that fl-laetoglobulin isolated by alcohol fractionation and differential solubility at p H 4.8 and 5.3 from a pooled nfilk supply showed a single eleetrophoretic peak in buffers alkaline to the isoelectric point and two maxima in buffers on the acid side. They isolated the slow-moving fraction ( p H 4.8) in small yields, which they designated fl~-lactoglobulin. The fast-moving component, which was not purified, was termed fl2-1aetoglobulin. Aschaffenburg and D r e w r y (4) prepared fl-laetoglobutin-rieh fractions from the milk of individual cows, which they studied by paper electrophoresis in veronal buffer at p H 8.6, r / 2 = 0.05. They observed that individual animals gave milk containing one or the other or a mixture of both of two electrophoretically discernible fl-lactoglobulins. They designated the faster of the two components fl~-laetoglobulin and the slower as fi_~-lactoglobulin. Ogston and Tombs (38), working with fl-lactoglobulin crystallized from the milk o'f individual cows, found that fll-laetoglobulin, the fastest-moving component on filter paper electrophoresis at p H 8.6, was also the fastest component observed during free-boundary electrophoresis in acetate buffer at p H 4.6. This observation caused them to regard the protein isolated by Polls as a subfraction, not related to the fl~-laetoglobulin observed by Asehaffenburg and D r e w r y (5). In addition, they observed that both fl~- and fl2-1actoglobulin were electrophoretically heterogeneous at p H 4.6. Klostergaard and Pasternak (23) observed that the mobility of Polls' fl~-laetoglobulin was slower than Aschaffenburg's fll-lacto-
TABLE 1 Protein fr~etlons of bovine skimmilk a n d some of their properties Protein fraction
(~lassical 11Olll e n c l a t u r e
~'
Contemporary n onlenela ture
(?t,sein (precipitated f r o m skimnfilk by acid at p H 4.6) a-casein
I n casein p a t t e r n (17) 1
Reference to prepar~tion
Approxim a t e per cent of skimmilk protein ~
Sedimentation constant ~ (S.-o)
18,58
76-86
1.18 (20) ~
15,0(210(20) 33,600 (6)
18, 58
45-63
3.99 (50)
]?] f
Electrophoretie mobility at, pH 8.6 ~
27,000 (36)
4.1 (22)
--6.7 (22)
Contains 1% phosphorus. Consists of a mixture of interesting proteins (see Table 2). :Formed hi the udder"
Molecular weight"
Other eharacteristies
fl-casein
2
18, 58
19-28
1.57 (50)
24,100 (50)
4.5 (22)
--3.1 (22)
0.6% Phosphorus. Formed in udder
3"-casein
3
18
3-7
1.55 (35)
30,600 (35)
5.8-6.0(22)
--2.0 (22)
0.1% Phosphorus. Preformed f r o m blood
2.8 (53)'
35,000 (as)
-5.3 (54)
Associates in p H range 3.7 to 5.3. Formed in udder
5.3 (53) J 2.7 (53)
35,000 (38)
-5.2 (54)
Exists principally in monomerie form. Formed in udder.
1.75 (15)
16,500 (15)
Noncasein proteins Laetalbumin (Soluble in saturated MgSO~ sohL)
Occurrence in electrophoretle pattern" (Peak No.)
]In acid whey p a t t e r n (27)
14-24
fl-Laetoglobulin A
6
4
fl-Lactoglobulin B (Mixed A and B)
6 39
4
a-Laetalbumin
4
7-12 h 15, 16
2-5
5.1 (24)'
-4.2 05)'" 7% tryptophane. Formed in udder
Blood serum albumin
7
42
0.7-1.3
4.0 (11)
69,000 (42)
4.7 (42)
-6.7 (42)
A p p a r e n t l y identieal to bovine serum albunfin. P r e f o r m e d f r o m blood
Laetoglobulin (Insoluble in saturated MgSO,~ soln.)
Euglobulin
1
48
0.8-1.7
8.77 (35) k
Pseudoglobulin
2
48
0.6-1.4
8.07 (35) k
3, 22, 27
2-6
0.96 (3) k 2.75 (31)
Proteose-Peptone Fraction (Not precipitated at pH 4.6 from s k i m m i l k preyiously heated to 95 100 ° C., 30 rain.)
3 5 8
1.0' ( 2 2 )
252,00t) 180,000 289,000 180,000
(35) k (48) k (35) k (48) ~
6.0 (35)
--1.8 (35)
5.6 (35)
2.0 (35)
4,90(~ (3) k 24,0O0 (3) --3.0 (27) --4.6 (27) k 7.9 (27)
F r a c t i o n s c o n t a i n i n g antibodies. C o n t a i n hexose a n d h e x o s a m i n e . Elect r o p h o r e t i e a l l y a n d uIt r a e e n t r i f u g a l l y heterogeneous. P r e f o r m e d f r o m blood. Glyeoprotein ( 5 1 ) . Elect r o p h o r e t i c a l l y a n d ult r a c e n t r i f u g a l l y heterogeneous. P o o r l y defined e x c e p t f o r s e r u m c o m p o n e n t 5 (21).
" R o w l a n d f r a c t i o n s (22, 46, 47). h l,'ree-boundary eleetrophoresis in vel'onal buffer at p H 8.6, F / 2 -- 0.1. Casein c o m p o n e n t s d e s i g n a t e d in d e s c e n d i n g order of mobility. S e r u m p r o t e i n c o m p o n e n t s d e s i g n a t e d in a s c e n d i n g order of mobility. " Values compiled a n d / o r c a l c u l a t e d f r o m R o w l a n d n i t r o g e n d i s t r i b u t i o n data, relative a r e a s of eleetrophoretie p a t t e r n s , a n d p r o t e i n yiehl studies (18, 22, 27, 45, 46, 47). a s=,, = s e d i m e n t a t i o n c o n s t a n t = ( d x / d t ) (1/w-~x), in S v e d b e r g m i l t s (S = ] × ] 0 "~) corrected to 20" C. See original l i t e r a t u r e for experim e n t a l conditions. Sedimentatim~ c h a r a c t e r i s t i c s are d e p e n d e n t u p o n ionic s t r e n g t h of solvent, t e m p e r a t u r e , p H , a n d c o n c e n t r a t i o n of solute. The s e d i m e n t a t i o n a n d molecular w e i g h t values reported are n o t necessarily t h e best w d u e s o b t a i n a b l e , nor do t h e y c o n s t i t u t e e n d o r s e m e n t by the Committee. ~' R e f e r to original l i t e r a t u r e f o r m e t h o d a n d conditions of d e t e r m i n a t i o n . f ]soeleetric point, i.e., p H of no eleetrophoretic m o v e m e n t . E l e e t r o p h o r e t i c mobility (u) = × 10 ~, era. ~, volts-J, see. -z o b t a i n e d by the Tiselius m o v i n g b o u n d a r y m e t h o d at 2 ° C. in v e r o m d lmffer a t p H 8.6, I " / 2 - = 0 . ] . N[e2~sured f r o m d e s c e n d i n g p a t t e r n . ~' I)istrilml.i(m (~f /~-lacloglolndill A :lml B ~lrc geJletic:flly d e t e r m i n e d ( 4 , 5 ) . Denotes tim ehar~eteristic of the m e n o m e r i e specie. J I lenotes l h e characteristic of the associated specie. k l)enol:es tile c h a r a c t e r i s t i c s of t h e n m j o r eonlt)onen{. Value replaces prcviously r e p o r t e d w d u e of 4.] to 4.8. "' Mobility r e p o r t e d at - - 3 . 6 in milk s e r m n p r o t e i n m i x t u r e (26). " Source of i n f o r m a t i o n p e r t a i n i n g to the origin of the milk p r o t e i n s (25).
TABLE 2 Reported f r a c t i o n s or components of a-casein and some of their properties"
C o n t e m p o r a r y nomenclature
Occurrence in electrophoretic pattern (Peak No.)
Reference to p r e p a r a tion
Approximate p e r cent of skiumlilk protein
Sedimentat a t i o n COils t a n t (S.2,,)
. Molecular weight
37-54 (61)
1.59(61) ~
23,300(59)
PI
Electrophoretle mobility at p H 8 . 6
Other characteristics
a-casein ~' Ca-sensitive c o m p o n e n t ( s )
(l)
as-casein
61 ~
~ -casein
33 ~
3.0 (34)
an-casein
30 ~
4.55(30)
Ca-insensitive component(s)
1.10% p h o s p h o r u s 4.3-4.7 (34)
--6.7(33)
0.85% p h o s p h o r u s 1.16% p h o s p h o r u s
( 1) (49,57,61)
~-casein
13,61 a'~
a.o-or a~-casein
33 °
a3-casein
19 ~
X-casein
30 ~
m casein
59, 60 f
]1-]3
1.4 (61) g 13.5 (61) h
0.19 to 0.33% p h o s p h o r u s StabiIizes a~-casein to Ca ions
16,300(59)
-5.0(33)
Stabilizes al-easein to Ca ion.
23.0 (].9) 1.2 (30)
1.18% p h o s p h o r u s , stabilizing.
1.1 (30)
" U n i t s and experimental conditions similar to those described for Table 1. " Method of isolation f r o m whole casein can determine content of calcium-sensitive components e Similar characteristics s u g g e s t t h a t p r o t e i n s are similar. ,1 Gross calcium-insensitive fraction. o Similar characteristics s u g g e s t t h a t p r o t e i n s are similar. Similar characteristics suggest t h a t p r o t e i n s are similar. Denotes characteristics of monomeric form. h Denotes characteristics of associated form.
0.1 to 0.15% p h o s p h o r u s . I s o l a t e d in small yields. Soluble at p H 4.7. Resembles ~-casein.
(6°).
not
908
J . R. B R U N N E R
E T AL
globulin on paper eleetrophoresis at p H 8.6, indicating that Pelts' fi~-laetoglobulin corresponded to Aschaffenburg's //~. This observation has since been confirmed in free-boundary eleetrophoresis by Timasheff and Towncnd (54). Asehaffenburg and D r e w r y (5) discovered that the secretion of fit-laetoglobulin and/32-1actogiobulin was under genetic control. This induced them to abando'u the old nomenclature in favor of the fl-laetoglobulin-A and B nomenelature which is more acceptable from a genetic standpoint. /?-laetoglobulin A and B are discernible in descending order of eleetropho'retic mobility in veronal buffer at p H 8.6. Recent work of Timasheff and Townend (52, 53, 54, 55) eontributed ~ignifieantly to the characterization of fl-laetoglobulins A and B. The electrophoretie mobility at p H 4.65 of B-lactoglobulin B was slower than the polymer Type A, but slightly faster than the monomer. They concluded, from supporting ultracentrifugal data, that the observed association of B-lactoglobulin in the p H range of 3.7 to 5.2 was due to fl-laetoglobulin A. Type B sedimented as a single molecular specie at concentrations up to 7%. The eom, lusions supported the work of Ogston and Tombs (38, 56), who suggested that the asso~'iation of /?-laetoglobulin was due to the aggregation or association of Type A. The following recommendations relative to the nomenclature of the fi-laetoglobulins are suggested: fl-laetoglo~bulin exists in two forms which are genetically defined and discernible by paper eleetrophoresis at p i t 8.6. These were designated by Asehaffenburg and D r e w r y (5) as fi-laetoglobulin A (faster-moving component) and B whieh appears to be the accepted designation for reasons lnentioned earlier, fi-laetoglobulin A associates in the range of p H 3.7 to 5.2, while /?-laetoglobulin B exists principally in a monomeric state. This particular characteristic of the fl-laetoglobulins manifests itself in the heterogeneity observed in the eleetrophoretic and ultracentrifugal patterns of fi-lactoglobulin A and B (mixed) and fi-laetoglobulin A, when studied witlfin this p H range. The fi-lactoglobulins are essentially monomeric in alkaline media. The foregoing disdussions serve as backgro'und material upon which the deliberations of this Committee were based. The present status of the available knowledge relating to the eharaeteristies of the protein fractions of milk presently known as ,-casein and fi-tactoglobutin has induced us to suggest revisions to the report o'f the 1956 Committee (see Tables 1 and 2). Obviously, the voids in data within these tables demonstrate the paucity of our information relating to physical and chemical properties of skimmilk proteins. Also, tha Committee wishes to point out a eertain lack of evidence in support of the homogeneity of fractions or components listed under a-casein, and that inclusion in the table does not eonstitute unmitigated aeeeptanee of the eomponent as a protein entity, but, rather, a compilation of the current information relatino' to the eharacteristies and nomenclature of the a-casein fraction. In some instam:es, apparently, similar components have been designated differently by the various autho'rs. The Committee hopes that by compiling these data, it willl, to ,-ome degree, stinmlate continued studies toward the elueidation of the milk l,rote'in system
NOMENCLATURE OF PROTEINS Free-boundary
909
e l e c t r o p h o r e s i s c o n t i n u e s to be r e c o g n i z e d as a p r i m a r y
a r d f o r classification in the n o m e n c l a t u r e
s c h e m e , b u t i t is a p p a r e n t
stand-
that other
p h y s i c a l a n d c h e m i c a l c r i t e r i a a r e r e q u i r e d t o e l u c i d a t e t h e c o m p l e x i t y o f electrophoretieally homogeneous components. REFERENCES
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