Zone Electrophoresis of Carbohydrates*

ZONE ELECTROPHORESIS* OF CARBOHYDRATES

BY A. B. FOSTER Chemistry Department, The University of Birmingham, England

I. Introduction ........................................................... 81 11. Technique of Zone Electrophoresis. ................... 1. Apparatus for Zone Electrophoresis. ............... a. The Suspended-strip Method. .................................... 84 b. The Immersed-strip Method.. ............................... 84 c. The Enclosed-strip Method.. .............................. 111. Zone Electrophoresis of Carbohydrates in the Presence of Borate. ...... 86 1. Neutral Carbohydrates.............................................. 89 a. Monosaccharides.. ................................................ 89 b. Methylated Sugars.. .. ..................................... 92 c. Di- and Oligo-sacchari ..................................... 95 d. Glycopyranosides ..................................... e. Glycofuranosides ..................................... f . Polyhydric Alcohols.. ............................................ 102 g. Flavonoid Glycosides. .......... ............................... 104 2. Acidic and Baaic Carbohydrates and Related Substances.. ..... 104 IV. Zone Electrophoresis of Carbohydrates in the Presence of Comp Agents Other than Borate. ............................................ 106 V. Determination of Molecular Size of Carbohydrates by Zone Electro107 phoresis.. .............................................................. VI. Zone Electrophoresis of Carbohydrates on Glass Paper. ........ VII. Zone Electrophoresis of Polysaccharides. ............................... 110 1. Acidic Polysaccharides.. ........................................ 2. Neutral Polysaccharides.. . . VIII. Separations of Carbohydrates on Ion-exchange Resins. ................. 114

I. INTRODUCTION The scope of paper' and column2chromatography in carbohydrate chemistry is now well defined, and the impact of these techniques has been widely felt. Complementary to chromatography, and at present developing rapidly, is zone electrophoresis. It may be stated that, whilst chromatography has

* Zone electrophoresis is the most precise name for the technique. Other terms in common use are ionophoresis, ionography, electromigration, and electrochromatography. (1) G. N. Kowkabany, Advances i n Carbohydrate Chem., 9,303 (1954). (2) W. W. Binkley, Advances i n Carbohydrate Chem., 10,55 (1955). 81

82

A. B. FOSTER

reached its majority, zone electrophoresis is currently adolescent, although its potentialities have been fully recognized. The general technique of electrophoresis may be classified conveniently into boundary electrophoresis and zone electrophoresis.3 Boundary electrophoresis, which is a well-established analytical method, involves the migration of charged molecules in free solution under the influence of an applied electric field. The migration of the components of a mixture may be observed by means of optical methods that utilize the differences in density at the boundaries between the solution of the migrating substances in the electrolyte and the electrolyte itself. Precautions must be taken to eliminate convection currents in the conducting solution, and, because of diffusion effects, the technique is usually applied to molecules of relatively high molecular weight, especially proteins. Complete separation of the components of a mixture is not normally achieved in boundary electrophoresis. In zone electrophoresis, the conducting solution and the migrating substances are supported on a solid matrix. By means of this device, convection effects are effectively eliminated; diffusion effects are, in general, counterbalanced by the high mobilities of the migrating substances attendant on the application of high voltage gradients; and it is usual for the components of a mixture to be separated into discrete zones. Location of the zones on the supporting matrix may be accomplished by making use of specific chemical reactions or physical properties of the migrating substances. The molecular weight of the migrating substance in zone electrophoresis is not necessarily so critical a factor as often is the case in boundary electrophoresis. I n fact, zone electrophoresis has been successfully applied to mono-, oligo-, and poly-saccharides and to an analogous range of molecules in the protein field. In the realm of carbohydrates, the majority of investigations have been concerned with mono- and oligo-saccharides. A comparative account of the application of boundary and zone electrophoresis to proteins has been given by Tiselius and F10din.~The development and application of zone electrophoresis and allied techniques in the carbohydrate field will be the main concern of this Chapter. Isolated examples of the use of zone electrophoresis have long been on record: but not until 1952 were the potentialities of its application to problems of carbohydrate chemistry realized generally. The development and wide application of zone electrophoresis in the amino acid-peptideprotein (3) A. Tiselius and P. Flodin, Advances i n Protein Chem., 8,461 (1953). (4) See, for example, C. W. Field and 0. Teague, J. E x p t l . Med., 9, 86 (1907); J. Kendall, E. R. Jette and W. West, J . Am. Chem. Soe., 48,3114 (1926); T. B. Coolidge, J . B i d . Chem., 127, 55 (1939). Current interest appears to have stemmed from the work of R. Consden, A . H. Gordon and A. J. P. Martin, Biochem. J. (London), 40, 33 (1946) and subsequent papers.

ZONE ELECTROPHORESIS OF CARBOHYDRATES

83

field3 is, perhaps, riot surprising, since the molecules there encountered possehs a nct charge or may be given one by c*ontrollitigthe pE-1 of their environnient. On the other hand, most members of the carbohydrate family arc elcctrically neutral. This, the major obstacle, was overcome by making use of the long-known reaction between borate ions and carbohydrates in aqueous solution, a reaction which leads to the formation of negatively charged complexes that migrate in an applied electric field. It cannot be over-emphasized that the techniques of chromatography and zone electrophoresis, which are based on very different physicochemical principles, are compkmentary; and, in those cases where both may be applied, a more powerful analytical method obtains than when either technique is used alone. Especially is this so in the field of carbohydrate chemistry.

11. TECHNIQUE OF ZONEELECTROPHORESIS Recently, detailed accounts of the methodology of zone electrophoresis have appeared,3*6 so that only the salient points of the technique need be mentioned here. The supporting matrix most commonly used in zone electrophoresis is filter paper, although such other substances as starch, silica, powdered glass, silica gel, and even single cellulose fibers, have found some applicationlo and may be more advantageous than paper in certain cases (see page 109). I n essence, zone electrophoresis involves the application of an electric field across a strip of filter paper17impregnated with a suitable conducting solution, onto which a suitable amount of the substance (5-150 pg.) under examination has been introduced as an arbitrarily located, compact zone. After a suitable interval of time, the paper strip is processed in much the same manner as in standard paper-chromatographic procedure. The position of the zones may be ascertained by making use of specific physical properties (for example, ultraviolet absorption) or chemical reactivity (for example, reducing power) of the migrated substances. Many of the detecting reagents which have been developed in connection with the chromatography of organic compounds are equally useful in zone electrophoresis. An extensive compilation of these detection reagents has been described by Block, Durrum and Zweig.6(*) (5) (a) R. J. Block, E . L. Durrum and G. Zweig, “A Manual of Paper Chromatography and Paper Electrophoresis,” Academic Press Inc., New York, N. Y., 1955. (b) M. Lederer, “Introduction t o Paper Electrophoresis and Related Methods,” Elsevier, Amsterdam, 1955. (c) H. J. McDonald, “Ionography,” The Year Book Publishers Inc., Chicago, Ill., 1955. (6) I,. F. J. Parker, Analyst, 80, 638 (1955). (7) All subsequent references t o zone electrophoresis imply the use therein of filter-pitper strips, unless otherwise stated.

84

A. B. FOSTER

1. Apparatus for Zone Electrophoresis In view of the fact that filter paper is the supporting matrix by far the most frequently used in zone electrophoresis, only those types of apparatus which employ filter-paper strips will be mentioned. Since, in zone electrophoresis, the impregnated, filter-paper strip is the effective resistance in an electrical circuit, heat will be generated within its structure. Ineffective or incomplete dissipation of this heat will lead consecutively to evaporation of the conducting solution, establishment of a concentration gradient, and, ultimately, the breakdown of the circuit if a dry zone is formed across the paper strip. The various types of apparatus in current use may be classified according to the devices employed to control the development of heat within the paper strip or to facilitate its dissipation. a. The Suspended-strip Method.-This technique involves the simplest, and possibly the least expensive, type of equipment. The impregnated or in an inverted paper strip may be suspended horizontally,8 ~ertically,~ V shapelo with the ends dipping into the electrode chambers. Precautions are usually taken to prevent polarized electrolyte (from the electrode chambers) from reaching the ends of the paper strip. The whole apparatus may be enclosed in a humidified chamber in order to minimize evaporation of liquid from the paper strip. Because of the largely inefficient cooling system (air convection), relatively low potential-gradients only may be applied, especially when conducting solutions of high ionic strength are being used. This restriction is reflected in the considerable intervals of time which may be found necessary in order to achieve satisfactory separations. b. The Immersed-strip Method.l'--In this type of apparatus, the filterpaper strip is immersed in a solvent which is immiscible with the conducting solution. In conjunction with an aqueous conducting solution, carbon tetrachloride or heptane may be used. The cooling is mainly effected by liquid convection and is more effective than in (11, la), thus permitting the use of higher voltage-gradients. c. The Enclosed-strip Method.l-It is the author's opinion that this type of apparatus, originally devised by Kunkel and Tiselius,12(8)is the most versatile of the three noted. Essentially, the apparatus comprises a filter(8) W. Grassmann andK. Hannig, Hoppe-Seyler's Z. physiol. Chem., 2 W , l (1952); see Ref. 5(a), p. 366. (9) H. Michl, Monatsh., 82,489 (1951). (10) E. L. Durrum, J . A m . Chem. SOC.,73,2943 (1950). (11) J. D. Smith and R. Markham, Nature, 168,406 (1952); E. L. Durrum, unpublished method, described in Ref. 5(a), p. 359. (12) (a) H. G . Kunkel and A. Tiselius, J . Gen. Physiol., 36, 89 (1951). (b) A. B. Foster, Chemistry & h&?tr?/, 1050 (1952).

ZONE ELECTROPHORESIS OF CARBOHYDRATES

85

paper strip enclosed between glass plates; the ends of the paper strip project beyond the edges of the glass plates, and dip into the electrode chambers. The plates are clamped together and onto a metal cooling-plate. Cooling is effected by conduction through the glass plates and it is surprisingly efficient in most instances. Enclosing and cooling the paper strip in this manner effectively prevents evaporation and permits the use of relatively high potential-gradients. Cooling may also be effected by immersing the clamped glass plates in a bath of ch10robenzene.l~Alternatively, the employment of low voltage-gradients or of conducting solutions of low ionic strength, or both, may obviate the need for a special cooling-device. With high potential-gradients and with conducting solutions of high ionic strength, the heat generated within the paper strip may be too great for rapid dissipation if glass plates are used. This difficulty may be overcome by substituting polythene sheets for the glass plates and by clamping them between two metal co~ling-blocks.~~ Two advantages may be gained by the use of high potential-gradients in zone electrophoresis. Firstly, and obviously, a rapid separation of the components of a mixture may be permitted and this will be of considerable importance in routine work. Secondly, diffusion effects (which may interfere with the resolution of mixtures of substances having closely similar mobilities) may be eliminated. I n fact, certain resolutions may be effected only under very high potential gradients.16 In practice, in designing equipment for routine use, a balance must be sought between the magnitude of the potential gradient employed and the associated electrical hazard. A convenient procedure12(b)for preparation of the paper strip is as follows. Solutions of the substances under examination are introduced onto the paper strip as compact zones along the origin line (which is a t right angles to the length of t h e strip and arbitrarily located). One end of the paper strip is then immersed in the conducting solution until the liquid front is about 1 cm. from the origin line. The impregnated portion is thoroughly blotted by inserting between two sheets of absorbent paper and strongly compressing with a rubber roller. The second portion of the paper is treated likewise; the two liquid fronts are then equidistant from, and near to, the origin line. By this procedure, t h e zones of the substances to be migrated are compactly retained on the origin line. The paper strip is inserted between the glass plates and the apparatus is assembled. The blotting procedure ensures tha t no liquid will be extruded from the paper strip when i t is compressed between the glass plates. A suitable potential-gradient is applied, and, after a n appropriate interval of time, the paper strip is processed by standard chromatographic methods. (13) H. D. Cremer and A. Tiselius, Biochem. Z . , 320, 273 (1950).

(14) D. Gross, Nature, 172,908 (1953). (15) D. Gross, Nature, 176,362 (1955).

86

A. B. FOSTER

Several strips may be used simultaneously if they are arranged in sandwich fashion and are separated by polythene sheets. The number of papers which may be so used will be limited by the amount of heat to be dissipated, which, in turn, will depend upon the thickness of the paper strips, the ionic strength of the conducting solution, and the potential gradient applied. Kunkel and Tiselius12(s)recommend that the glass plates, which enclose the paper strip, should be covered with a film of silicone grease in order to eliminate any anomalous movement at the paper-glass interface. This precaution is unnecessary if the paper strip is prepared as described above. The possibility that some of the substances in the migrating zones might be lost by transference to the glass plates was ruled out when it was shown that, after zone electrophoresis of a nucleic acid hydrolyzate, the only ultraviolet-absorbing material transferred to the glass plates originated from the paper itself and not from the migrating nucleic acid components.16 Thus, the enclosed-strip technique may be used for quantitative work.

111. ZONE ELECTROPHORESIS OF CARBOHYDRATES IN THE PRESENCE OF BORATE which showed that, in In 1952, a number of publications the presence of alkaline borate, a wide variety of neutral carbohydrates migrate toward the anode in zone electrophoresis. It is of interest to note that Coleman and Miller,22ain 1942, had observed the migration of D-glucose and maltose toward the anode when a potential was applied across their solution in aqueous borax. The reaction of polyhydric alcohols with borate ions has long been knownz3and it has been suggestedz4that the following equilibria occur.

(16) A. B. Foster, A. S. Jones and S. B. H. Rizvi, Chemistry & Industry, 914 (1956). (17) R.Consden and Winifred M. Stanier, Nature, 169,783 (1952). (18) Y. Hashimoto, I. Mori and M. Kimura, Nature, 170,975 (1952). (19) H. Michl, Monatsh., 8s, 737 (1952). (20) L. Jaenicke, Naturwissenschaften, 39, 86 (1952). (21) L. Jaenicke and P. Vollbrechthausen, Naturwissenschaften, 39,86 (1952). (22) A. B. Foster, Chemistry & Industry, 828 (1952). (22a) G. H. Coleman and A. Miller, Proc. Iowa Acad. Sci., 49, 257 (1942). (23) See C. A. Zittle, Advances i n Enzymol., 12,493 (1951). (24) J. Boeseken, Rec. trav. chim., 61,82 (1942). See also, H. S. Isbell, J. F. Brewster, Nancy B. Holt and Harriet L. Frush, J . Research Natl. Bur. Standards, 40, 129 (1948). See also, Ref. 40.

ZONE ELECTROPHORESIS OF CARBOHYDRATES

Lo~o.l” + HO

HO

OH

\

87

R &

HO’

HO

[ H o x l > R I

+

I

HO

R, \

~

HO I1

Presumably, it is the ionic species I and I1 which migrate in zone electrophoresis. Since the equilibria (l), (2), and (3) are dynamic, then, over a sufficiently long period of time, all the carbohydrate molecules R(OH), will be associated with a negative charge. The magnitude of this charge will be determined by the position of the equilibria ( l ) , (2), and (3), which will be influenced, among other things, by the stereochemical disposition of the hydroxyl groups in R(0H)z (see p. 98). The concentration of the ionic species I and I1 in aqueous boric acid is low, and an increase in pH would be expected to raise their concentration and, concomitantly, to result in an increased electrophoretic mobility of the polyhydric alcohols. That this is indeed the case is clearly demonstrated by the results of Consden and Stanier.” Figs. 1 and 2 show the mobility-pH relationship for a range of simple carbohydrates. Two important inferences may be drawn from Figs. 1 and 2. (1) The maximum mobilities of the carbohydrates are generally in the pH region 9-10; and, consequently, borate buffers within this range are usually selected for zone electrophoresis. (2) The relative mobilities of certain pairs of carbohydrates may be critically dependent on pH; for example, at pH 9-10, D-glucose has a mobility greater than that of D-fructose, whereas, at pH 7-8, the reverse relationship obtains. Thus, careful selection of an appropriate pH may be of value in facilitating certain separations. Consden and Stanierl7 noted that, at high pH values, the migrated zones were sharp and circular, but that, a t lower pH values, “some were rather elongated, suggesting the existence of more than one species of complex.” A t pII 7 , only the ketoses and n-ribose had any appreciable mobility and, in boric acid (pH 5 ) , the migrated zones of these sugars became elongated. A further significant observation” was that, at pH 8, the mobility of carbohydrates is proportional to the borate content of the buffer, suggesting that, under these conditions, some of the carbohydrates in solution are uncomplexed.

88

A. B. FOSTER

The M a value has been suggested12(b)v 26 as a convenient index of the mobility of a carbohydrate in zone electrophoresis. The M Q value, which bears a formal resemblance to the RQ value employed in chromatography, is given by

MQ=

true distance of migration of a substance true distance of migration of D-glucose

-

-

PH PH FIG. 1.-Relationship Between Mobility and pH, for Glucose (l),Galactose ( 2 ) , Fructose (3), Ribose (4), and Raffinose ( 5 ) . FIG. 2.-Relationship between Mobility and pH, for Sorbose (l),Arabinose ( 2 ) , Mannose (3), Rhamnose (4), and Cellobiose ( 5 ) .

The true distances of migration are obtained by correcting for movement due to electroendosmotic flow. With borate buffers, the electroendosmotic flow is toward the cathode, so that the true distances of migration are usually greater than the apparent distances. Movement occasioned by electroendosmotic flow is determined by referring to the position of substances which do not react with borate ions, for example, 2,3,4,6-tetra-O-methylD-glucose12(b)and trans- 1 ,2-~yclohexanediol.~~ For a given system of zone electrophoresis, the M o values are fairly reproducible, and they will be used (25) A. B. Foster, J . Chem. Soc., 982 (1953). (26) A. B. Foster, unpublished observation.

ZONE ELECTROPHORESIS OF CARBOHYDRATES

89

in this Chapter wherever possible. It has been inferred2'. 28 that there is little or no selective absorption of low molecular-weight carbohydrates (that is, hexasaccharides and smaller oligosaccharides) on paper during zone electrophoresis. Several observations support this inference; thus, di$erent substances which do not complex with borate ions, for example, methyl a-D-xylopyranoside, 2,3,4,6-tetra-O-methyl-~-glucose, and trans1,2-cyclohexanediol, migrate at identical rates under the influence of the electroendosmotic flow. Further, in a glycine buffer at pH 11, members of the oligosaccharide series maltose-maltohexaose, and also the a- and P-Schardinger dextrins, all migrated to the same extent under the influence of the electroendosmotic flow?* Elimination of the observed slight variations in M , values for a given zone-electrophoretic system is experimentally very difficult, since it would demand precise control of (1) the quality and thickness of the paper strips, (2) the buffer content per unit volume of paper, (3)the pressure exerted on the paper strip by the enclosing medium, (4)the amount of carbohydrate in each migrating zone, and (5) the temperature. For most practical purposes, these variations are not very important, since a comparison between known and unknown compounds is performed on a single, paper strip. 1. Neutral Carbohydrates

In the following Sections, the various types of carbohydrate which have been subjected to zone electrophoresis have been classified arbitrarily. I n view of the fact that zone electrophoresis is, as yet, relatively little used in carbohydrate chemistry, an attempt has been made to emphasize and illustrate the applicability of the technique and to compare it, at the appropriate point, with paper chromatography, rather than to compile a catalog of applications. a. Monosaccharides.-Most of the early investigations on the zone electrophoresis of carbohydrate~17-~0 were concerned, in the main, with monosaccharides. A comparative list of M , and R p values of a range of monosaccharides is shown in Table I, from which it may be seen that there are significant differences in the movement of carbohydrates in zone electrophoresis and in paper chromatography (see also, Table 11).For example, D-galactose, D-glucose, and D-mannose may be easily separated by zone electrophoresis, but not readily by paper chromatography. It is important to note that separations achievable by both techniques will, in general, be much more rapidly accomplished by zone electrophoresis. It is also clear from Table I that, in zone electrophoresis, carbohydrates of different (27) E. J. Bourne, A. B. Foster and P. M. Grant, J. Chem. SOC.,4311 (1956). (28) A. B. Foster, Primula A. Newton-Hearn and M. Stacey, J. Chem. SOC.,30

(1956).

90

A. B. FOSTER

“types,” for example, pentoses and hexoses, may have identical or closely similar M , values ; specific examples are the pairs D-xylose-D-glucose and L-arabinose-D-galactose. This observation suggests the manner in which zone electrophoresis and paper chromatography may be used complementarily in the identification of carbohydrates or in substantiating the homogeneity of individual components of a mixture. Separation of a mixture of carbohydrates into fractions of different types may be achieved by paper chromatography or by column chromatography. Elution of the fractions TABLE I Comparative MG and RF Values of Some Monosaccharides R p i n solvent systema Sugar

L-Arabinose D-Ribose D-Xylose L-Fucose L-Rhamnose D-Galactose D-Glucose D -Mannose D-Fructose L-Sorbose L-galacto-Heptulose n-manno-Heptulose

h!

0.96 0.77 1 .oo 0.89 0.52 0.93 1.00 0.72 0.90 0.95 0.89 0.87

1

2

3

4

0.43 0.56 0.50 0.44 0.59 0.34 0.39 0.46 0.42 0.40 -

0.21 0.31 0.28 0.27 0.37 0.16 0.18 0.20 0.23 0.20 -

0.51

0.12 0.21 0.15 0.21 0.30 0.06 0.082 0.11 0.12 0.10 0.11

-

-

0.34 0.59 0.56 0.35 0.29 0.35 0.45 0.36 -

-

a 1. Water saturated with 2,4, 6-collidineBe 2. 1-Butanol-acetic acid.29 3. Phenolacetic acid-water.80 4. l-Butanol-ethanol-water.81

from the paper chromatogram or from the column, and subjecting them to zone electrophoresis, will in many cases indicate homogeneity or identity. This sequence of operations was employed to advantage by Gross32in an investigation of the products of the action of yeast invertase on sucrose. A further example is provided by R i ~ k e t t s , who * ~ observed that the product obtained on treatment of a dextran sulfate with aqueous alkali gave, on acidic hydrolysis, a mixture of reducing sugars which migrated as a single zone in paper chromatography. The Rp value was similar to that of D-glucose, but more than one substance appeared to be present. On treatment (29) S. M. Partridge and R. G. Westhall, Biochem. J. (London), 42,238 (1948). (30) J. N. Counsell, L. Hough and W. H. Wadman, Research (London), 4, 143 (1951). (31) E. L. Hirst and J. K. N. Jones, Discussions Faraday Soc., 7, 268 (1949). (32) D. Gross, Nature, 173,487 (1954). (33) C. R . Ricketts, J . Chem. SOC., 3752 (1956).

ZONE ELECTROPHORESIS OF CARBOHYDRATES

91

by zone electrophoresis, four discrete zones were obtained, with M , values identical to those of D-glucose (1.00), D-gulose (0.84),D-altrose (0.92), and D-mannose (0.72). The rapid separations of carbohydrates by means of zone electrophoresis may be of value in studying the course of a reaction. Thus, Stacey and coworkers34were able to follow the conversion of D-glUCa1 and of D-galactal into 2-deoxy-D-‘(glucose” and Z-deoxy-D-“galactose,” respectively, under the influence of a cation-exchange resin (He form). The reaction was shown to be quite complex. An indication of the rapidity of zone electrophoretic separations is shown by the fact that, using the enclosed-strip technique12(b)with a borate buffer a t pH 10 under a potential gradient of about 20 v./cm., a mixture of maltose and isomaltose may be completely resolved in 20 minutes. Under similar conditions, a mixture of D-glucose and D-galactose would be separated in about 4 hours. Most routine separations may be effected in time periods lying between these two limits. I n the interests of time saving, it should be noted that, by using specially designed equipment, potential gradients of up to 240 v./cm. may be applied to a 50-cm. paper strip.36 Many proteins contain small amounts of bound polysaccharide, and paper-chromatographic analysis of the hydrolyzates of these substances (for carbohydrates) is rendered difficult by the excess of amino acids present. The isolation of carbohydrate-rich fractions may be a n essential preliminary to hydrolysis. These difficulties may be overcome to a large extent by zone electrophoresis, which frequently will permit the separation of sugars from amino acids.” In a borate buffer at pH 8.6, only the “peptides of glutamic acid and aspartic acid will migrate to positions similar to those of the monosaccharides,” but, even so, they are usually present in small proportions. The separated sugars may be examined subsequently by paper chromatography and, if necessary, by zone electrophoresis again. The general movement of sugars in zone electrophoresis is slowed down by the presence of amino acids and peptides. Consden and Stanier” have employed a single paper sheet, first for zone electrophoresis and second for paper chromatography. They used the following general method. The protein polysaccharide is hydrolyzed at 100-110” for 3 hours with N sulfuric acid, neutralized with baryta solution, filtered, and the filtrate concentrated t o small volume. Zone electrophoresis in borate buffer (pH 8.6)is then carried out for a suitable time, and the paper strip is dried and sprayed with ninhydrin30 (to locate the amino acids) and with aniline hydrogen phthalate37 (to detect the reducing sugars).

(34)A. S.Matthews, W . G. Overend, F. Shafizadeh and M. Stacey, J . Chem. Soc., 2511 (1955). (35)D. Gross, Nature, 178, 29 (1956). (36) Ref. 5(a), p. 88. (37) S. M. Partridge, Nature, 164, 443 (1949).

92

A. B. FOSTER

The relative positions of the amino acids and sugars may give useful information. Alternatively, the paper may be subjected t o chromatography in a direction a t right angles to that of the zone electrophoretic separation; in which case, the paper should finally be sprayed first with aniline hydrogen phthalate and then with ninhydrin.

Using this procedure, Consden and Stanierl’ were able to show that a hydrolyzate of Group A hemolytic streptococci contained hexosamine, rhamnose, ribose, and glucose; and, also, that a hydrolyzate of purified, human fibrin contained uronic acid, hexosamine, mannose, and galactose. A similar combination of techniques has been employed by Woodin38in a study of the composition of a corneal mucopolysaccharide. It is of interest that the conditions of acidic hydrolysis necessary for releasing the D-glucosamine moiety from heparin also result in destruction of the concomitantly released D-glucuronic acid.agTraces of the intact uronic acid may be revealed by zone electrophoresis of the hydrolyzates.26 b. Methylated Sugars.-The behavior of methylated sugars under zone electrophoresis was first described by Foster,22who found that mixtures of 2,4- and 3,4-di-O-methyl-~-rhamnose, which are difficult to resolve by paper chromatography, are easily separated by zone e1ect)rophoresis (see Table 11) since only the latter ether can form a complex with borate ions. This behavior was predicted from the work of Boeseken,4O who inferred that, for cyclic carbohydrates, only vicinal cis-hydroxyl groups can form a complex with borate ions in aqueous boric acid. The observation**that 2,3-di-O-methyl-~-glucose, the furanose and pyranose forms of which have no vicinal cis-hydroxyl groups, has an MQ value of 0.12 suggested that, in alkaline media, other types of borate-ion interaction could occur. The M Qvalues listed in Table I1 indicate that this is indeed the case (RQ values are included for purposes of comparison). Several of the derivatives listed, especially those of D-glucose substituted at C2, show an appreciable mobility on zone electrophoresis in alkaline borate, although the furanose and pyranose forms have no vicinal cis-hydroxyl groups. A further important observation is the low MQ value of 4-0-methyl-~-glucose;in this case, the furanose form is precluded, but the a-D-pyranose form has vicinal cishydroxyl groups at C1 and C2. The M Q values of the mono-0-methyl-& glucoses suggest that the hydroxyl groups on C2 and C4 in the parent sugar are very important in complex formation with borate ions. It is possible that the important role of the hydroxyl group at C4 is associated with a reaction of the furanose form with borate ions; however, other results (see page 101) suggest that the furanose form may not be significantly involved (38) A. M.Woodin, Biochem. J . (London), 61,319 (1952). (39) M.L.Wolfrom and J. V. Karabinos, J. A m . Chem. Soc., 67,679 (1945). (40) J. Boeseken, Advances i n Carbohydrate Chem., 4, 189 (1949). (41) A. B.Foster and M. Stacey, J . Appl. Chem. (London), S,l9 (1953).

93

ZONE ELECTROPHORESIS OF CARBOHYDRATES

in complex formation. The sequence of the M Qvalues in Table I1 has been rationalized by postulating that the aldehydo form of the sugars is the principle one involved in complex formation.41aIn this event, the pair of hydroxyl groups sterically most favorable for complex formation are those on C2 and C4. It must be admitted, though, that little or no information is available which would indicate how the equilibria of furanose, pyranose, TABLEI1 MQ and RF V a l u e s of Some Methylated, Acetamidodeoxy and Deoxy Sugars Sugar derivative

D-Glucose 2-0-Methyl-~-glucose ‘‘~-D~OX~-D-~~UCOB~” 2-0-Methyl-~-galactose “2-Deoxy-~-galactose” 2-Acetamido-2-deoxy-~-glucose 2-Acetamido-2-deoxy-~ -galac tose 3-O-Methyl-~-glucose 4-O-Methyl-n-glucose 4-O-Methyl-~-galactose 6-O-Methyl-~-glucose 2,3-Di-O-methyl-~-glueose 2,4-Di-O-methyl-~-ghcose

3,4-Di-O-methyl-~-glucose 2,3,4-Tri-O-methyl-~-glucose 3,5,6-Tri-O-methyl-~-glucose 2,3,4,6-Tetra-O-methyl-~-ghcose 2,3-Di -0-methyl-L-rhamnose 2,4-Di-O-methyl-~-rhamnose 3,4-Di-O-methyl-~-rhamnose

RQ

Mg26.41

1.00 0.23 0.29 0.32 0.37 0.23 0.35 0.80 0.24 0.27 0.80 0.12 <0.05 0.31 0.00 0.71 0.00 <0.05 <0.05 0.36

in solvent“ B

0.09 0.22 0.23 0.25 0.26 -

-

0.27 0.57 0.52 0.85 1.oo 0. 832a 0. 872a 0.892’

0.69*’ 0. 6441 0. 6541

a Solvent A is 1-butanol-ethanol-water.Solvent B is l-butanol-ethanol-waterammonia.

and open-chain forms of reducing sugars in alkaline media are influenced by complex formation with borate ions. A limitation of zone electrophoresis in the field of methylated sugars is the non-reaction of several polymethylated derivatives with borate ions, but, as the results in Table I1 testify, in the mono- and di-0-methyl series there is a much wider variation in Mg values than in Ro values. Bell and Northcote& have reported the mobilities of the 0-methyl-Dfructoses given in Table 111. (41a) See H. Bouveng and B. Lindberg [Acta Chem. Scand., 10,1283 (1956)l for the application of this theory to the mono-0-methyl-D-gdactoses. (42) D. J. Bell and D. H. Northcote, Chemistry & Industry, 1328 (1954).

94

A. B. FOSTER

Zone electrophoresis has found application in the structural investigation of 2,4-di-O-methyl-~-rhamnose,isolated from a methylated polysaccharide from Pneumococcus Type 1143and from okra mucilage,44and of 2,4-di-O-methyl-~-glucoseisolated after methylation and hydrolysis of a dextran elaborated by Betacoccus a r a b i n o s a c e o ~ s The . ~ ~ behavior of several mono- and di-0-methyl ethers of D-ribose in zone electrophoresis has been been ~tudied.~0. 47 It was found47that exhaustive methylation of uridylic acid “b” with the Purdie reagents, followed by hydrolysis, gave, after paper chromatography, a di-0-methyl-D-ribose fraction which was shown, by zone electrophoresis, to contain two components corresponding to 2 , 5 and 3,5-di-O-methyl-~-ribose. Evidently, phosphate migration had ocTABLE I11 Mobilities of O-Methyl-D-fructoses42 D-Fructose deriwalive

Dislance migrated‘ i n cm.

1-0-Methyl 3-0-Methyl3,4-Di-O-methyl4,5-Di-O-methyl1,3,4-Tri-O-methyl1,4,5-Tri-O-rnethyllJ4,6-Tri-0-methyl3,4,6-Tri-O-methyla The mobility of D-fructose itself was not recorded. pH 9.2. c Used a8 a marker.

14.7 14.0 10.8 13.0 0.00” 9.7 12.5 9.3 Borate buffer (0.05 M ) of

curred during the methylation. Application of zone electrophoresis to the product obtained by the action of diethylmagnesium on methyl 2,3-anhydr0-4,6-O-benzylidene-cu-~-mannoside, followed by acidic hydrolysis, helped to substantiate its identity as a mixture of 3-deoxy-3-C-ethyL~altrose and its 1,6-anhydro d e r i ~ a t i v e . ~ ~ The examples selected above illustrate some of the many possible applications of zone electrophoresis. (43) K. Butler, P. F. Lloyd and M. Stacey, Chemistry & Industry, 107 (1954). (44) R. L. Whistler and H. E. Conrad, J . Am. Chem. SOC.,7 6 , 3514 (1954). (45) S. A . Barker, E. J. Bourne, G. T. Bruce, W. B. Neely and M. Stacey, J . Chem. Soc., 2395 (1954). (46) D. M. Brown, G. D. Fasman, D. I. Magrath and A . R. Todd, J . Chem. SOC., 1448 (1954). (47) D. M. Brown, D. I. Magrath and A . R. Todd, J . Chem. Soc., 1442 (1954). (48) A. B. Foster, W. G. Overend, M. Stacey and G. Vaughan, J . Chem. SOC.,3308 (1953).

95

ZONE ELECTROPHORESIS OF CARBOHYDRATES

c. Di- and Oligo-saccharides.-From the zone-electrophoretic behavior of the mono-0-methybglucoses, it might be predicted that the reducing disaccharides of D-glucose (only) with (1 -+ 2) or (1 -+ 4) linkages would have much lower M Qvalues than those containing a (1 + 3) or a (1 -+ 6) linkage. The M , values in Table IV amply confirm this prediction: maltose and cellobiose have much lower mobilities than have isomaltose and gentiobiose. Furthermore, certain pairs of disaccharides which contain linkages at similar positions (but of different configuration) have appreciably different M , values, for example, maltose and cellobiose, and isomaltose and gentiobiose. The migration of di- and oligo-saccharides in paper chromatography with the customary solvent systems is very slow, but special solvent systems have been evolved which result in much higher RQ values.49Al-

TABLEIV MG Values of Some Disaccharidese6 Disaccharide (DGkbCOSyl-D-gllUOSC)

Sophorose Nigerose Laminaribiose Maltose Cellobiose Lactose Isomaltose Gentiobiose

Linkage

P-D-(1

-+

2)

CU-D-(l + 3)

P-D-(l -+ 3) a-D-(l 4) D-D-(1 -+ 4) p-D-(1 + 4) -+

C U - D - ( ~+ 6)

p - ~ - (+ 1 6)

0.24 0.69 0.69 0.32 0.23

0.38 0.69 0.75

ternatively, the di- or oligo-saccharides may be converted, on the paper, to a suitable derivative (for example, the N-benzylglycosylamine60)which will have a greater solubility in the mobile phase and hence a higher RF value. It has been emphasizedzs that a combination of these chromatographic procedures with zone electrophoresis may be of considerable value in the identification of di- and oligo-saccharides. The most important structural feature which governs the magnitude of the M Qvalue of a reducing D-gluco-oligosaccharide (and probably of many other saccharides), is the point of attachment of the “remainder” of the molecule to the reducing moiety. The M Qvalue will be influenced to a much smallcr extent by structural variations in the “remainder” of the molecule. An interesting example of this phenomenon was encountered in structural investigations of the polyglucan elaborated by Aspergillus niger.61The poly(49) Allene Jeanes, C. S. Wise and R . J. Dimler, Anal. Chem., 23, 415 (1951). (50) R . J. Bailey and E. J. Bourne, Nature, 171, 385 (1953). (51) S. A . Barker, E. J. Bourne and M. Stacey, J . Chem. Soc., 3084 (1953).

96

A. B. FOSTER

saccharide contains an alternating sequence of a-~-(l-+ 3) and C X - D4 - ( ~4) links, and graded acidic hydrolysis afforded a trisaccharide fraction (by carbon-Celite column chromatography) which appeared homogeneous by paper chromatography using the benzylamine method,60 but which had an R, value intermediate between that of a (1 + 3) ,(1 4 3) linked trisaccharide and a (1 + 4) ,(1 -+4) linked trisaccharide. Zone electroph~resis~~ of the trisaccharide fraction gave two components with the mobilities expected for (1 -+ 3), (1 -+ 4) and (1 4 4), (1 -+ 3) linked trisaccharides. Use has been made of zone electrophoresis in structural investigations of glycogen. Peat, Whelan and Edwards62isolated, by carbon-Celite column chromatography, a trisaccharide fraction from a partially hydrolyzed glycogen from baker’s yeast. Zone electrophoresis showed two components, of low and high mobility. Extraction of the slow-moving component from the paper, followed by reduction (with NaBH4) and acetylation, gave panitol dodecaacetate, indicating the parent trisaccharide to be panose [an a - ~ - ( l-+ 6) , a - ~ - (-+l 4)-linked trisaccharide of D-glucose (only)]. Thelow mobility of this trisaccharide is not unexpected. It was suggested62 that the component of high mobility might be the a - ~ - ( + l 4) , c x - D - ( ~+ 6)linked trisaccharide. Recently, Wolfrom and Thompson63fractionated a partial hydrolyzate of glycogen by carbon-Celite column chromatography, paper chromatography, and zone electrophoresis, successively. A trisaccharide fraction was obtained which had a mobility much higher than that of panose. Structural investigation showed it to be isomaltotriose, and not the (1 -+ 4) ,(1 + 6)-linked trisaccharide which might have been expected. This significant observation reveals that some of the (1 -+ 6) links in glycogen must be in adjacent positions. The preceding examples elegantly demonstrate the point at which zone electrophoresis may be used to advantage in structural determinations on polysaccharides, namely, in further resolving fractions isolated by carbonCelite column chromatography or paper chromatography, or both. Recovery of sugars from “pherograms”64 (paper electrophoretograms) impregnated with sodium borate involves extraction with water followed by the removal of inorganic material from the extracts. This may be accomplished by removing cations with a suitable ion-exchange resin, followed by evaporation of the solution and the removal of boric acid as the volatile methyl borate by repeatedly distilling added methanol from the residue.66 (52) S.Peat, W.J. Whelan and T. E. Edwards, J . Chem. SOC.,355 (1955). (53) M.L.Wolfrom and A. Thompson, J . Am. Chem. Soc., 78, 4182 (1956); 79, 4214 (1957). (54) T. Biicher, D.Matselt and D. Pette, Klin. Wochschr., 30,325 (1952). (55) Compare L.P.Zill, J. X. Khym and G. M. Cheniae, J . Am. Chem. SOC.,76, 1339 (1953).

ZONE ELECTROPHORESIS OF CARBOHYDRATES

97

I n the case of di- and oligo-saccharides, a simpler method may be employed. The aqueous extract of the pherogram is added to a short carbon-Celite column, inorganic material is eluted with water, and the carbohydrate is subsequently eluted with aqueous ethanol.26This method may be used for any substance which is not readily eluted from a carbon-Celite column by water but which can be eluted with aqueous ethanol. Alternatively, the saccharide extracted from the pherogram may be a c e t ~ l a t e d . ~ ~ Special precautions may be necessary in the recovery of saccharides which include a (1+ 3) link at the reducing end of the molecule. These saccharides are readily decomposed by alkali.66It is interesting to note that laminaribiose and nigerose are stable under the conditions (borate pH 10) of zone electrophore~is,2~ but are decomposed on extraction from the pher~gram.~’ In this case, it may be necessary to render the pherogram neutral before performing the extraction. Isolation, using zone electrophoresis, of a quantity of an oligosaccharide sufficient for structural studies may be a tedious process, and it may be necessary to prepare and then extract many pherograms. An important, column technique has been developed68which will overcome this difficulty in many cases. The method is based on the well-known, carbon-Celite colunin technique of Whistler and D u r s ~ The . ~ ~column, prepared in the usual manner, is impregnated with a borate buffer (pH lo), and the eluant (aqueous ethanol) contains this buffer at the same p H . Saccharides which complex strongly with borate ions are eluted more rapidly from the column than would be the case in the absence of borate. The reason for this behavior lies in the fact that the borate complexes have some of the characteristics of inorganic salts, and it is well known that many salts are not firmly retained b y carbon-Celite columns. Thus, of a pair of isomeric saccharides, the one complexing the more strongly with borate ions will be the more rapidly released from the column. For example, a mixture of melibiose and maltose (with M , values of 0.80 and 0.32, respectively) is incompletely resolved on a normal, carbon-Celite column when a gradient of aqueous ethano160 is applied; the disaccharides emerge when the eluant contains 3.2 % and 5.3 % of alcohol, respectively. Using the borate-impregnated column, resolution is complete, and the melibiose emerges a t 0.8% and the maltose a t 4.6% concentration of alcohol. It should, therefore, be feasible, in many cases, to translate a microscale, zone-electrophoretic (56) Compare W. M. Corbett and J. Kenner, J . Chem. Sac., 3274 (1954). (57) S. A. Barker, E. J. Bourne and M. O’Mant, unpublished observations, cited in Ref. 58. (58) S. A. Barker, E. J. Bourne and 0. Theander, J . Chem. Sac., 4276 (1955). (59) R. L. Whistler and D. F. Durso, J . A m . Chem. Sac., 7 2 , 677 (1950). (60) B. Lindberg and B. Wickberg, Acta Chem. Scand., 8,569 (1954).

98

A.

€3.

FOSTER

separation to a macroscale separation, using a borate-impregnated carbonCelite column. Information on the feasibility of such separations will be obtainable from the relative M u values. TABLE V MG Values of Some Pento- and Hero-pyranosides and Related Compoundse1-B3 Glycoside

Methyl a-D-xylopyranoside j3 anomer lt5-Anhydroxylitol Methyl a-D-arabinopyranoside j3 anomer 1,5-Anhydro-~-arabinitol Methyl a-D-lyxopyranoside p anomer Methyl 8-D-ribopyranoside 1,5-Anhydroribitol Methyl a-D-glucopyranoside fl anomer 1,5-Anhydro-~-glucitol Methyl a-D-galactopyranoside j3 anomer 1,5-Anhydro-~-galactitol Methyl a-n-mannopyranoside 8 anomer 1,5-Anhydro-~-mannitol Methyl a-L-rhamnopyranoside j3 anomer 1,5-Anhydro-~-rhamnitol Met hy1 a m - gu 1opy ranosi de j3 anomer Methyl a-D-fructopyranoside fl anomer Sucrose Trehalose, a,aa,@-

838-

0.00 0.00 0.00 0.38 0.38 0.39 0.45 0.27 0.53 0.53 0.11 0.19 0.20 0.38 0.38 0.38 0.42 0.31 0.40 0.31 0.14 0.31 0.59 0.72 0.71 0.59 0.17 0.19 0.23 0.19

d. G2ycopyranosides.-The zone-electrophoretic behavior of a wide range of glycopyranosides has been studied,26zm3and the M , values are recorded in Table V. The zero M u values of D-xylopyranosides indicate that vicinal, trans-hydroxyl groups do not react with borate ions. This is in accord with the observations of Boeseken using boric acid.40It is of interest to note that , (61) A. B. Foster, E. F. Martlew and M. Stacey, Chemistry & Industry, 825 (1953). (62) A. B. Foster and M. Stacey, J . Chem. Soc., 1778 (1955). 1395 (1957). (63) A. B. Foster, J . Chem. SOC.,

ZONE ELECTROPHORESIS OF CARBOHYDRATES

99

whilst the anomeric methyl D-arabinopyranosides and 1,5-anhydro-~arabinitol have similar affinities for borate ions, this is not the case with the anomeric methyl D-lyxopyranosides. Methyl a-D-lyxopyranoside has a much higher M Qvalue than has the p anomer. I n fact, in all the examples presented in Table V where vicinal, cis-hydroxyl groups are flanked by a cis related methoxyl group (that is, in methyl 8-D-lyxopyranoside, methyl P-L-rhamnopyranoside, methyl p-D-mannopyranoside, and methyl a-D-gulopyranoside), the reaction of the hydroxyl groups with borate ions appears to be hindered. Although a precise explanation of this effect cannot be given a t present, it seems probable that the relative instability of the borate complexes of these glycosides, resulting in low M , values, is due in part t o adverse, non-bonded interactions within the complexes. In this regard, it would be instructive to determine the M , value of methyl a-D-ribopyranoside. The mobilities of methyl a- and 0-D-glucopyranosides in zone electrophoresis are attributable to the formation of a borate complex across the hydroxyl groups at C4 and C6. This has been shown by blocking individual hydroxyl groups in derivatives of these glycosides. An explanation of the lower mobility of methyl a-D-glucopyranoside (O.ll), compared to that of the p anomer (0.19), may be offered in terms of non-bonded interactions. The borate complexes of the a and ,f3 anomers may be represented by 111 and IV, which show the complexes to be rigid, trans fused, bicyclic systems. The glycosidic methoxyl group in the borate complex of methyl p-D-glUCOpyranoside (IV) occupies an equatorial position, where it is relatively free from strong non-bonded interactions. In the complex of the a anomer (111), however, the glycosidic methoxyl group occupies a n axial position and will interact strongly with the axial hydrogen atoms on C3 and C5. This effect will tend to destabilize the borate complex of the a anomer and is reflected in the lower M a value.

OH

OH

I11

1v

Sugihara and P e t e r ~ e nhave ~ ~ shown that metaboric acid will condense with methyl a-D-glucopyranoside under suitable conditions. Benzoylation (64) J. M. Sugihara and J. C.Petersen, J . A m . Chem. Soc., 78, 1760 (1956).

100

A. B. FOBTER

of the condensate, followed by methanolysis of the boric ester groups and acetylation of the liberated hydroxyl groups, yielded a mixture of products in which there predominated the 4,6-diacetate-2,3-dibenzoate and the 3,4-diacetate-2,6dibenzoate of the original glycoside. Since the acetyl groups substituted the hydroxyl groups which had originally carried the boric ester groups, it appears that 4,6- and 3,4-borate esters are formed in the initial condensation. As noted above, a borate-ion interaction at the hydroxyl groups at C4 and C6 has been observed in the zone electrophoresis of methyl a-D-glucopyranoside. No evidence has been obtained which would indicate-the formation of a complex across the hydroxyl groups at C3 and c4. Both of the steric effects mentioned above are operative in the D-mannopyranosides. Thus, in methyl a-D-mannopyranoside, the 4,6-borate complex is hindered, but the 2,3-c0mplex is not. The reverse situation obtains for the 8 anomer. Since the 2,3-c0mplex is formed more readily than the 4,6-complex, the a anomer has the greater M , value. The similar M , values of the anomeric methyl D-galactopyranosides and the anomeric methyl D-arabinopyranosides indicate that their reactions with borate ions are independent of configuration at the glycosidic center, It is the occurrence of borate-ion interactions, similar to those found for methyl a- and 8-D-glucopyranoside, that is responsible for the mobility of sucrose and the trehaloses in zone electrophoresis. The structures allocated on the basis of the reaction of the anomeric methyl D-fructopyranosideswith borate ions (as reflected in the M , values) are consistent with the structures allocated on the basis of their optical rotations.03It is interesting to note that methyl a-D-fructopyranoside (V) and methyl P-D-gulopyranoside (VI), which have a closely related disposition of their hydroxyl groups, have closely similar M , values. CHzOH

H

H

HO V

VI

e. G1ycojuranosides.-From the M , values of the anomeric methyl D-arabinofuranosides and methyl D-xylofuranosides,it may be inferred that, whereas vicinal, cis, hydroxyl and hydroxymethyl groups (as in the, D-xylofuranosides) in these glycosides react strongly with borate ions, a trans arrangement of these groups (as in the D-arabinofuranosides) permits a

101

ZONE ELECTROPHORESIS OF CARBOHYDRATES

very weak interaction. Thus, the structures allocated to the anomeric methyl D-fructofuranosides, on the basis of their M , values, are the same as those allocated on the basis of their optical rotations.aaA knowledge of this type of borate-ion interaction has been of value in structural investigations on 2,5-anhydro-~-mannito1,66and in interpreting the M , values of the adenosine 2-, 3-, and 5-phosphate~.~~, 66 Together with a knowledge of the borate-ion interactions noted in Section 111, Id, the (observed) mobility relation kestose (a-~-Gp-(l+ 2)-P-~-Fruf-(6+ 2)-P-~-Fruf)> neokestose (P-~-Fruf-(24 6)-a-~-Gp-(l4 2)-p-~-Fruf)would have been predicted. The isomeric trisaccharides kestose and neokestose were obtained by the action of yeast invertase on sucrose,67and this investigation provides TABLEV I Me Values of Some Glycofuranosides and a Related Cornpound62*68 Dnivafioe

Methyl a-D-arabinofuranoside B anomer Methyl a-D-xylofuranoside j3 anomer Methyl a-D-fructofuranoside 13 anomer Methyl a-D-glucofuranoside 1,2-O-Isopropy~idene~-~-glucofuranose Methyl a-D-galac tofuranoside j3 anomer

0.035 0.035 0.56 0.33 0.60 0.04 0.73 0.73 0.41 0.31

yet another example of the value of zone electrophoresis in the resolution of mixtures of isomeric saccharides. The high M , value of the D-glucofuranoside derivatives in Table VI is due to complex formation with the hydroxyl groups at C3, C5, and C6, and is of considerable interest in connection with the role of furanose forms in the reaction of reducing derivatives of D-glucose with borate ions (see page 93). The low M o value of 2-O-methyl-~-glucose,in which the hydroxyl groups at C3, C5, and C6 are free would suggest that the furanose form of this sugar is not significantly involved in complex formation. It is not improbable that the hydroxyl groups a t C3, C5, and C6 in the D-glucofuranoside derivatives shown in Table VI may form a “ tridentate complex” of the type suggested for certain inositol derivativesas (see page 104). (65) B.C.Bera, A. B. Foster and M. Stacey, J. Chem. SOC., 4531 (1956). (66) D.C.Burke and A. B. Foster, Chemistry & Industry, 94 (1955). (67) D.Gross, P.H. Blanchard and D. J. Bell, J. Chem. Soe., 1727 (1954). (68) S.J. Angyal and D. J,McHugh, Chemistry & Industry, 1147 (1956).

102

A. B. FOSTER

f. Polyhydric Alcohols.-Using a borate buffer at pH 9.2, Frahn and Millss3have studied the zone-electrophoretic behavior of a number of diols (see Table VII). Thus 1,4-butanediol and 1 ,5-pentanediol were found not to react with borate ions, indicating that borate complexes involving 7- and 8-membered rings have little tendency to be formed. The more ready reaction of threo-2 ,3-butanediol with borate ions, in comparison with the erythro isomer, is not unexpected, since the 5-membered ring in the borate complex of the latter (but not the former) would possess eclipsed methyl groups a t C2 and C3. The relative reactivity of the 2 ,3-butanediols toward borate TABLEVII MG Values of Some Pol?/hydric Alcohols 27.09 Derivative in solvent" A

1,4-Butanediol 1,5-Pentanediol threo-2,3-Butanediol erythro-2,3-Butanediol 2,4-Pentanediol 1,3-Pentanediol Glyceritol Erythritol u-Arabinitol Galactitol u-Glucitol D-Mannitol

0.00 0.00 0.56 C.14 0.00, 0.35 0.05, 0.19

0.76 0.92 0.83

in solvenP B

0.44 0.75 0.90 0.98 0.89 0.90

Solvent A is borate buffer of pH 9.2. Values of M a are calculated from the d a t a of Frahn and Mills.69 Solvent B is borate buffer27 of p H 10.

ions is closely analogous to the reaction of acetone with threo- and erythro1 ,2-di-C-phenyi-l , 2-ethanediol.'o The stereochemical implications involved in the cis cyclization reactions, of which the above are examples, have been discussed by Barton and Cookson.71Although it is understandable that 2,4-pentanediol should yield two components on subjection t o zone electrophoresis, it is difficult to see why 1,3-pentanediol should behave in a similar manner, since the two possible forms of the diol are enantiomorphs and should have a n equal affinity for borate ions. Gross16 has pointed out that, until the advent of zone electrophoresis, (69) J. L. Frahn and J. A. Mills, Chemistry & I n d u s t r y , 578 (1956). (70) P. H. Hermans, 2. physik. Chem., 113, 337 (1924). (71) D. H. R . Barton and R. C. Cookson, Quart. Revs. (London), 10, 44 (1956).

ZONE ELECTROPHORESIS OF CARBOHYDRATES

103

there was no satisfactory micromethod for the separation of the hexitols shown in Table VII. The zone electrophoretic separation of galactitol, D-glucitol, and wmannitol is complicated by the fact that their mobilities lie within a iiarrow range. Attempts to resolve a mixture of the hexitols at low potential-gradients may be complicated by the normal diffusion of the migrating zones (an effect which will tend to cause the zones to overlap). The diffusion effects can be minimized sufficiently to permit a satisfactory resolution of the mixture by operating a t high potential-gradients.15 Gross was also able to separate these three hexitols from D-glucose, D-fructose, D-mannose, and L-sorbose. The mobility sequence of the hexitols may be critically dependent on the conditions of zone electrophoresis, since, a t pH TABLEV I I I Ma Values of Some Inositols72, 73 Inosilol ~

~~

myo-Inositol (+)-Inositol (-)-Inositol scyllo-Inositol epi-Inositol do-Inositol muco-Inositol Quebrachitol Pinitol Bornesitol Sequoyitol

I

MQ

0.53 0.63 0.63 <0.05 0.73 0.88 0.96 0.31 0.66 0.12 0.18

9.2, Frahn and Mills69 observed the mobility sequence of D-glucitol > D-mannitol > galactitol, whereas GrossI6 (using pH 9.2) and Bourne, Foster and Grant2' (using pH 10) observed the sequence galactitol > D-mannitol > D-glucitol. The zone-electrophoretic behavior of a number of inositols has been studied,72p7 3 and the M , values listed in Table VIII indicate a wide range of mobilities within the group. Recently, Angyal and McHugh68have postulated the formation of a novel kind of borate complex with certain inositols. They observed that inositols with cis related hydroxyl groups at C1, C3, and C5 give complexes with borate ions even when cis-1 ,2-glycol groupings are absent. For example, scyllo-quercitol (VII) is postulated to yield the '' tridentate" complex VIII. (72) A. B. Foster and M. Stacey, Chemistry & Industry, 279 (1953). (73) A. B. Foster, Chemistry & Industry, 591 (1953).

104

A. €3. FOSTER

OH

aH I

OH

HO

HO

OH VII

H

VIII

The stability of the tridentate complex and, hence, the position of the equilibrium between the tridentate and the free inositol will be dependent on the stereochemical disposition of the hydroxyl groups not linked to the boron atom in VIII. The greater the number of these hydroxyl groups in axial positions, the less stable will be the complex. This effect is elegantly illustrated by the magnitude of the equilibrium constant in the following quercitol and inositol series (the number of free hydroxyl groups in the tridentate in axial positions is given in parentheses after the cyclitol): scyllo-quercitol (2) 5.0; epi-quercitol (1) 310; cis-quercitol (0) 7.9 x lo3; myo-inositol (2) 25; epi-inositol (1) 7.0 X lo3;and cis-inositol (0) 1.1 X lo6. As the number of axially disposed free hydroxyl groups in the tridentate decreases, the equilibrium constant rises sharply. Zone electrophoresis has been employed in structural investigations of the mono-0-methylinosito1s.~z-74 g. Flavonoid G1ycosides.-Relatively little attention has so far been paid to the zone electrophoresis of flavonoid glycosides. Hashimoto and coworkers1*have reported the migrated distances given in Table IX.

2. Acidic and Basic Carbohydrates and Related Substances The zone-electrophoretic behavior of a few carbohydrate acids has been reported. Foster and Stacey*' demonstrated the mobility sequence of D-glucuronic acid > n-galacturonic acid, in a borate buffer at pH 10. Consden and StanierI7 noted that uronic acids and hexosamines may be separated from neutral sugars in borate buffers of suitable p H . I n the author's opinion there is much scope for the application of zone electrophoresis in structural investigations of polysaccharides containing uronic acids. Gross36has studied the mobility of a range of nonvolatile organic acids in non-borate buf(74) L.Anderson and A. M. Landel, J . Am. Chem. SOC.,76,6130 (1954).

105

ZONE ELECTROPHORESIS O F CARBOHYDRATES

fers. He observed a fairly good correlation between the dissociation constants and the mobilities. A conducting solution at pH 2 (0.75 M formic acid) was found most satisfactory; at higher pH values there was a tendency to form multiple spots. The following mobility sequence was observed by Gross? gluconic acid < 2-ketogluconic acid < a-ketobutyric acid < a-ketoglutaric acid < glucosyl phosphate < glyceritol 3-phosphate < fructose 1,6-diphosphate. The most suitable use of borate buffers will be in the separations of mixtures of isomeric polyhydroxy acids. The behavior of phosphate, triose phosphate, and hexose phosphate under a variety of conditions in nonborate buffers was examined by Neil and Walker:6 who found that the Migration Distance2 Flauonoid Glycoside

TABLEIX f Some Flavonoid Cflycosidesla 'icinal cis-hydroxyl :roups in the sugar moiety

Vicinal hydroxyl groups in the aglycon

1

1 1 1 1

Myricitrin Rutin Quercitin Myrecetin Lutedin 7-glucoside Naringin Hesperidin Acacetin Morin Robinin

1

0 0

0 0 0 0 0 0

1 1 0 0 0 0

Migrationa distance

( t o cathode)

30 25 16 10 3 3 3 3 2 2

The buffer solution was 2% borax.

hexose monophosphates could not be separated from each other. These authors point out that an advantage of zone electrophoresis over chromatography is that useful fractionation of tissue extracts may be achieved directly. Separations of phosphates of certain isomeric carbohydrates are possible using borate buffers as shown by the mobility sequencedl: D-galactosyl phosphate > D-glucosyl phosphate, D-galactosyluronic acid phosphate > D-glucosyluronic acid phosphate. The speed of separation of acidic carbohydrates by zone electrophoresis is illustrated by the fact that, under suitable conditions,4l D-glucuronic acid may be separated from D-glucosyluroriic acid phosphate in 1.5 hours, whereas 4-6 days are necessary for effecting a resolution by paper chromatography. A considerable amount of attention has been focussed on the zone-electrophoretic behavior of components of nucleic acids, but this field has (75) N. W. Neil and D. G. Walker, Biochem. J . (London), 66, xxvii (1954).

106

A. B. FOSTER

recently been adequately reviewed by SmithT6and need not be further mentioned here. The separation of aminodeoxy sugars from neutral and acidic sugars may be readily achieved26using an acetate buffer at pH 5 (the N-acetyl derivatives of D-glucosamine and D-galactosaminehave characteristic M , value# in borate buffer at pH 10). M. C. Foster and A ~ h t o n ’have ~ found zone electrophoresis to be useful in the separation of streptomycin components. They observed the following mobilities ( M X lop5): streptomycin, 22.5; mannosidostreptomycin, 19.5; streptothricin, 24.0; streptidine, 24.9; and streptarnine, 6.3.

TABLEX Relative Migrations of Some Carbohydrates i n the Presence of Borate and Other Complexing A g e n t P Carbohydrate

D-Glucose D-Mannose D-Fructose D-Mannitol D-Glucitol Galactitol

I

I

Mobilitp Borax (1)

Arrenife (2)

17.0 11.7 15.1 14.1 15.7 12.8

1.0 2.4 5.1 7.5 6.3 9.3

I

Basic lead acetalc ( 3 )

0.7 4.5 2.8 2.8 3.6 4.6

a The figures quoted are the distances migrated (in cm.) under standard conditions. For the significance of solutions ( l ) , (2), and (3), see the text.

Although phenylhydrazones of carbohydrates have not been studied by zone electrophoresis, the behavior of phenylhydrazone derivatives of numerous a-ketoacids has been reported.78

IV. ZONE ELECTROPHORESIS OF CARBOHYDRATES IN THE PRESENCE OF COMPLEXING AGENTS OTHER THAN BORATE The zone electrophoresis of carbohydrates in the presence of complexing agents other than borate is a virtually unexplored field. Frahn and Mills69 have described some preliminary experiments in which the zone-electrophoretic behavior of carbohydrates in the following conducting solutions was compared: (1) borax, pH 9.2; (2) sodium arsenite-arsenious acid, pH (76) J. L). Smith, in “The Nucleic Acids,” E. Chargaff and J. N. Davidson, eds., Academic Press Inc., New York, N. Y., 1955, Vol. 1, p. 267. (77) M. C. Foster and G. C . Ashton, Nature, 172, 958 (1953). (78) W. J. P. Neish, Rec. t7an. chim., 72, 105 (1953); B. Mondori and F. Narazio, G i o ~ nbiochim., . 3, 259 (1954); H. Tauber, Anal. Chein., 27, 287 (1955).

ZONE ELECTROPHORESIS OF CARBOHYDRATES

107

9.6; and (3) basic lead acetate. In solutions (1) and (a), the carbohydrates migrated as anions; they moved as cations in solution (3). The rates of migration in solutions ( 2 ) and (3) were much lower than those in solutiorl (1). The relative migrations are summarized in Table X, from which it may be seen that the sequence of increasing migration distances is not the same for each conducting solution. Of especial interest is the behavior (in the arsenite buffer) of the hexitols, which, in addition to migrating more rapidly than the sugars, are more widely separated from each other than in borax (compare Gross16). I n conducting solutions containing sodium tungstate (pH 6.2) and ammonium molybdate (pH 5.6), D-mannitol, D-glucitol, and galactitol migrated rapidly as anions on subjection to zone electrophoresis, but were not separated. Reducing sugars also migrated, but extensive streaking occurred. The behavior of carbohydrates on carbon-Celite columns69impregnated with molybdate has recently been described,7gand comparisons were made with their behavior on borate-impregnated columns6s. Zone electrophore~is~~ of the hexitols in sodium metavanadate (pH 8.6) revealed the mobility sequence : D-glucitol > D-mannitol > galactitol.

V. DETERMINATION OF MOLECULAR SIZEOF CARBOHYDRATES BY ZONEELECTROPHOEESIS The fact that carbohydrates of different molecular sizes [for example, pentoses and hexoses (see Table I)] may have identical M , values on subjection to zone electrophoresis (in borate buffers) indicates that this system cannot be used for determining the molecular size of a carbohydrate. It has been emphasized a t several points in this Chapter that the most potent use for zone electrophoresis of carbohydrates in borate buffers is after mixtures of carbohydrates have been resolved into groups of similar molecular size by chromatographic procedures. Zone-electrophoretic methods have recently been developed whereby the molecular weight of an aldose may be ascertained. The first method, due to Stacey and coworkers,80involves the conversion of the aldose into the N-benzylglycosylamine on the pherogram (by interaction with benzylamine). Thereafter, the glycosylamine is caused to migrate as the glycosylammonium ion by zone electrophoresis in a formic acid-sodium formate buffer (pH 1.8). The mobilities listed in Table XI are expressed relative to that of N-benzyl-n-glucosylamine. It was shown that the mobility of N-benzylglycosylammonium ions is inversely proportional to the mo (79) S. A. Barker, E. J. Bourne, A . B. Foster and R . B. Ward, Nature, 179, 262 (1957). (80) S. A. Barker, E. J. Bourne, P. M. Grant and M. Stacey, Nature, 177, 1125 (1956).

108

A. B. FOSTER

lecular weight of the ions and is independent of the stereochemistry of the sugar and of the configuration of the linkages in di- and oligo-saccharides. Frahn and Millss1 have shown that zone electrophoresis of aldoses in TABLE XI Relative Mobilities of Aldoses, as N-Benzylglycosylammonium IonsaQ and as B k ite Complexess1

-

Aldose or derivative

liobilily o j Nieneylglycosylmmonwm ton, relative to that zf N-bcneyl-Dglucosylomine

Pentose

1.09-1.15

6-Deoxyhexose Hexose

1.oo

0.91

liol. put.

Aldose or derivative

Mobility of isul&c com$lex, relative to thot of D-glucose

150 150 150 164 180 180 180 206

Heptose tetra-0-methylhexose 4,6-0-benaylidenehexose Hexose disaccharide

0.71-0.78

Hexose trisaccharide

0.59-0.63

Hexose tetrasaccharide Hexose pentasaccharide Hexose hexasaccharide

0.49-0.51

504 666

0.42

828

0.33

990

210 236 268 342 342 342 342 342 504

D-xylose D-ribose D-arabinose L-rhamnose D-glucose D-galactose D-mannose 4,6-O-ethylidene-~-glucose

1.13 1.16 1.15 1.07 1.00 1.02 1.00 0.97

2,3,4,6-tetra-O-rnethyl-~

0.90

glucose 4, 6-0-benzylidene-~-glucose cellobiose maltose lactose melibiose isomaltose maltotriose panose maltotetraose

0.85 0.70 0.71 0.69 0.71 0.70 0.55 0.55 0.45

-

aqueous sodium bisulfite results in a separation according to the molecular weights. The technique involves the introduction of the aldose onto the pherogram, which has already been impregnated with 0.4M aqueous sodium bisulfite, and a suitable interval of time must be allowed in order to permit the formation of the bisulfite complex. During migration, the com(81) J. L. Frahn and J. T. Mills, Chemistry &Industry, 1137 (1956).

ZONE ELECTROPHORESIS O F CARBOHYDRATES

109

plexes undergo slow decomposition, to an extent dependent on the nature of the aldose. Under the conditions of zone electrophoresis described b y Frahn and Mills,s1ketoses did not afford a migrating component. The relative mobilities as determined by each method are listed in Table XI, from which it may be seen that they are related to the molecular weight; this permits the determination of the molecular size of an aldose, a t least up t o a hexasaccharide. The decrease of mobility with increase in molecular weight is not linear. Frahn and Millss1 applied the bisulfite method in a study of the acidreversion products of D-galactose. It was shown chromatographically that, after reversion, a t least five reducing compounds are formed in addition to n-galactose. After elution from the chromatogram, these components could be classified as di-, tri-, or tetra-saccharides by determining their zoneelectrophoretic mobility in aqueous sodium bisulfite.

VI. ZONEELECTROPHORESIS OF CARBOHYDRATES ON GLASSPAPER There are some disadvantages associated with the use of paper strips in the zone electrophoresis of carbohydrates ; for example, numerous nonreducing carbohydrates are difficult to locate after migration, and certain polysaccharides, especially the amylosaccharides, tend to be adsorbed (see page 113). To a large extent, these difficulties may be overcome by using strips fabricated of fiber-gla~s.2~. S2 Fiber-glass sheets of structure and properties suitable for zone electrophoresis are now available commer~ially.8~ Smith and coworkers838have obtained the most satisfactory results with the fiber-glass sheets made by the National Bureau of Standards a s recommended by O’Leary, Hobbs, Missimer and E r ~ i n g . 8 ~ ~ The chemical inertness of the fiber-glass sheets permits the application of vigorous chemical reactions for detecting zones of migrated carbohydrate. For example, 0.5 % potassium permanganate in N sodium hydroxide will detect sugars, methylated sugars, sugar alcohols, lactones, sugar phosphates, and neutral and acidic polysaccharides.s2 The more stable methylated methyl glycosides, methylated polysaccharides, and acetal derivatives may be detected by using 5 % 1-naphthol in 10 N sulfuric acid.s2The spray reagents are applied to the dried pherogram at looo, and may be applied successively to the same fiber-glass sheet. Smith and coworkers82 point out that certain quantitative determinations of carbohydrates may be simplified by the use of fiber-glass, since no contaminant carbohydrate can be (82) D. R. Briggs, E. F. Garner and F. Smith, Nature, 178,154 (1956). (83) Supplied by H. Reeve Angel and Co., Ltd., Bridewell Place, London, England. (83a) Prof. F. Smith, private communication to Prof. M. L. Wolfrom. (83b) M. J. O’Leary, R. B. Hobbs, J. K. Missimer and J. J. Erving, T a p p i , 37, 446 (1954).

110

A. B. FOSTER

extracted from the fiber-glass as may be the case with cellulose sheets. Furthermore, by using a suitable washing treatment, the fiber-glass sheets may be reclaimed for further use. Bourne, Foster and Grantz7have studied the zone-electrophoretic behavior of a wide range of simple carbohydrates on fiber-glass and cellulose sheets under similar conditions. It was observed that, whilst D-glucose has an appreciably lower absolute mobility on cellulose than on fiber-glass, the M , values of the range of compounds studied were only slightly different (in general, < 0.05),and it was concluded that there was negligible selective adsorption on either of these electrolyte supports. The electroendosmotic flow for fiber-glass was toward the cathode, but it was very much higher than with cellulose and the migrating zones tended t o become more diffuse on fiber-glass than on cellulose. As noted earlier, however, diffusion effects may be diminished by operating at high potential-gradients (page 103). It is the author’s opinion that the use of fiber-glass sheets will be of great value in studying the zone-electrophoretic behavior of polysaccharides.*

VII. ZONE ELECTROPHORESIS OF POLYSACCHARIDES 1. Acidic Polysaccharides

One of the earliest investigations of the zone-electrophoretic behavior of carbohydrates was concerned with the separation of mixtures of certain acidic poly~accharides.~~ BlixS6had shown that the boundary-electrophoretic mobilities of hyaluronic acid acd chondroitin hydrogen sulfate are quite different. Gardell, Gordon and Aqvista4examined the zone-electrophoretic behavior of these mucopolysaccharides, using 0.1 M acetate buffer and slabs of powdered silica or diatomaceous earth (Hyflo Supercel) as the electrolyte support. The latter support was found to be the more effective. The mucopolysaccharides were located by cutting the slab into strips, eluting with water, and determining the carbohydrate content of the eluate by a colorimetric method. In this manner, mixtures of chondroitin hydrogen sulfate and hyaluronic acid (20 mg. of each) could be resolved. The observed higher mobility of chondroitin hydrogen sulfate is to be expected, since the mucopolysaccharide contains sulfate groups, whereas the hyaluronic acid does not. The method was subsequentlys4applied to an extract of pig skin, and it was qualitatively demonstrated that two components, with mobilities

* Since this manuscript was completed, the zone-electrophoretic behavior of several neutral polysaccharides (including yeast mannan and snail galactan) on fiberglass strips (and on silk) has been described in detail by K. W. Fuller and D. H. Northcote [Biochem.J . (London), 64, 657 (1956)]. (84) S. Gardell, A . H. Gordon and S. i q v i s t , Acta Chem. Scand., 4,907 (1950). (85) G. Blix, Acta Physiol. Scand., 1, 29 (1940).

111

ZONE ELECTROPHORESIS OF CARBOHYDRATES

similar to those of hyaluronic acid and chondroitin hydrogen sulfate, are present. RienitsX6later studied the zone-electrophoretic behavior of hyaluronic acid, chondroitin hydrogen sulfate, and heparin on paper strips in nonborate buffers; he found that, whereas hyaluronic acid can be separated from chondroitin hydrogen sulfate and heparin, the latter two mucopolysaccharides cannot be separated. The mucopolysaccharides could be, located by the use of a method similar to that of Gardell, Gordon and A q v i ~ t , 8 ~ or by staining the pherogram with Toluidine Blue. The mobility of hyaluTABLEXI1 Mobilities of Some Neutral and Acidic Polusaccharidesas MobililyD

Polysaccharids

(Ir X 10-6 cm.?hec.-l/v.-l) ~~

Heparin N-(2,4-Dinitrophenyl)heparin Chondroitin hydrogen sulfate (from bovine trachea) (from bovine septa) Pneumococcus polysaccharide, Type I Type I1 Type I11 Rhizobium radicicolum polysaccharide Alginic acid Dextr an Amylose

-13.8 -13.5 -10.7 -11.9 -9.1 -3.4 -8.3 -9.9 -12.9

0.0 0.0

Determined in 0.06 M barbiturate buffer of pH 8.5. Detected by Toluidine Blue (acidic polysaccharides)

.

ronic acid was observed to be dependent on its state of polymerization; the more degraded samples had a higher mobility. The highly polymerized preparations of hyaluronic acid may have been adsorbed on the paper t o some extent. I n this connection, it has been reported that the mucopolysaccharides from thyroid follicle tend to be adsorbed on the paper in zone electroph~resis.~~ Rien itP applied his method t o extracts of pig skin, and concluded that several mucopolysaccharides appeared to be present. The mobilities of the polysaccharides in Table XI1 were obtained by Pasternak and Kentes using a 0.06 M barbiturate buffer at pH 8.5. (86) K. G. Rienits, Biochem. J . (London), 63,79 (1953). (87) G. J. Hooghwinkel, G. Smits and D. B . Kroon, Biochim. et Biophys. Acta, 16,

78 (1954). (88) C . A. Pasternak and P. W. Kent, Research (London)] 6,486 (1952).

112

A. B. FOSTER

2. Neutral Polysaccharides

Kent and coworkerssgbriefly reported the detection, after application of zone electrophoresisin barbiturate and other buffers, of a number of neutral polysaccharides, although no details of mobilities were noted. In a borate buffer at pH 8.0, amylopectin was observed to migrate toward the cathode and to be stained red with iodine, whereas amylose is stained blue and remains stationary. Preece and Hobkirkgohave attempted the fractionation of water-soluble, cereal polysaccharides (obtained from rye and oats), using zone electrophoresis on paper in a borate buffer at pH 11 and an acetate buffer at pH 4.The fractions were located on the pherogram by segmenting it, eluting TABLE XI11 Zone-electrophoretic Separation of Cereal PolysaccharideP Cereal

Buffer

Rye

borate

Oats

borate

Oats

acetate

a Key: tected.

Mooemenl

-+ cathode

stationary cathode stationary 4 anode --* cathode stationary + anode --f

1

I

I XylosP I Galaclose" I +++ ++ +

GlucosP

-I

f

+++ f

f

+++ -

-

+-

-

+-

-

++ ++ ++ ++ +++ ++ +++

Arabinosc?'

++

++ +++ +++ +++ +++ +++ +++

+++, major component; ++, minor component; +, trace; -, not de-

the segments, hydrolyzing the eluted carbohydrate, and then checking the sugars present by chromatography. The results shown in Table XI11 indicate that some fractionation was obtained; this permitted an interpretation of the composition of the original mixtures. In a detailed study of the zone-electrophoretic behavior of amylosaccharides on paper using a borate buffer (pH lo), Foster, Newton-Hearn and StaceyZ8showed that, whilst the amylopectin (blue value, 0.18) underwent appreciable migration toward the anode with but little adsorption of the polysaccharide in the path of movement, the pattern of movement of amylose (blue value, 1.35) depended on the amount of the polysaccharide put on the paper. Thus, a sufficiently small amount was adsorbed at the origin, but, with larger amounts, migration toward the anode occurred. A mixture of amylose and amylopectin was easily resolved, whereas, with (89) R. M. Greenway, P. W. Kent and M. W. Whitehouse, Research (London) 6 , 6s (1953). (90) I. A. Preece and R. Hobkirk, Chemistry & Industry, 257 (1955).

70NE ELECTROPHORESIS OF CARBOHYDRATES

113

phosphate and glycine buffers, little migration occurred and no separation could be achieved. Thus, it appeared that the migration both of amylose and amylopectin was due to the acquisition of a negative charge by complex formation with borate ions. As is known,62 methyl a- and @-D-glucopyranosides react with borate ions exclusively at the hydroxyl groups at C4 and C6, so that, in the amylosaccharides, which are linked CY-D-(~-i4), only the nonreducing chain-ends may react in this manner with borate ions. Since 4-O-methyl-~-ghcoseis known to react with borate ions (see Table 11),the reducing ends of the amylosaccharide chains may also react with borate ions. The contribution to the net negative charge of the borate complexes of amylose and amylopectin (from the reducing ends) would be expected to be the same, since there is only one reducing D-glucose unit per molecule. However, amylopectin has a much greater number of nonreducing chain-ends per molecule than has amylose, and glycogen has a still greater number. The observed mobility sequence: glycogen > ainylopectin > amylose is therefore to be expected. The probability of occurrence of other types of complex formation with borate ions, involving parts of the amylosaccharide molecules other than the end groups, must be considered. Both the a- and the p-Schardinger dextrin, in which all the hydroxyl groups at C4 are involved in glycosidic linkages, form complexes with borate ions. The Schardinger dextrins are well known for their ability t o form inclusion complexes with a variety of molecules, and it appears that a similar complex is formed with borate ions thereby conferring a negative charge on the cyclodextrins. The helical structure of the chains in amylose and amylopectin may similarly entrap borate ions. Foster, Newton-Hearn and Stacey28 provided examples of the application of zone electrophoresis to amylosaccharides in the presence of borate. thus, i t was possible to demonstrate some significant differences between amylose and amylopectin, on the one hand, and the synthetic amylosaccharides obtained by the action of P- and &-enzymes (from potatoes) on a-D-glucosyl ph~ sp h ate,~on' the other. The zone-electrophoretic behavior of the polysaccharide elaborated by Neisseria perJava was found to be unusual. Chemical investigationsgZhave revealed the polysaccharide to be a glycogen in type, but it has some rather unusual proper tie^.^^ On zone electrophoresis, it was found to be completely absorbed at the origin, whereas glycogens from other sources (ox liver, hog round-worm, bee-drone larvae, bass liver, human liver, sheep tapeworm, dog liver, frog liver, chicken liver, guinea-pig liverS*(b))all migrated toward the anode to a similar extent. (91) S. A. Barker, E. J. Bourne, S. Peat and I. A. Wilkinson, J . Chem. Soc., 3022 (1950); S. A. Barker, E. J. Bourne and I. A. Wilkinson, ibid., 3027 (1950). (92) (a) S. A. Barker, E. J. Bourne and M. Stacey, J . Chem. Soc., 2884 (1950); (b) M. Abdel-Akher and F. Smith, J . A m . Chem. SOC.,73,994 (1951). (93) S. A. Barker, A. Bebbington and E. J. Bourne, J . Chem. Soc., 4051 (1953).

114

A. B. FOSTER

These results emphasize the value of results obtained by the application of zone electrophoresis in the comparison of polysaccharides of supposedly similar chemical structure. An interesting study of the behavior of a range of neutral polysaccharides on subjection t o boundary electrophoresis in the presence of borate has been described by N o r t h c ~ t e . ~ ~ VIII. SEPARATIONS OF CARBOHYDRATES ON ION-EXCHANGE RESINS The technique of the separation of carbohydrates on a borate anion-exchanger was originated by Khym and Zill,96and a review of some of their results has been given by C ~ h nIt. is~ mentioned ~ in concluding this Chapter because of its relationship to zone electrophoresis. Briefly, the technique consists in eluting mixtures of carbohydrates from a column of strong-base resin (Dowex-1) by means of aqueous solutions of boric acid or sodium borate. Those sugars which react strongly with borate ions, thereby acquiring a high negative charge, will be more strongly sorbed by the resin than sugars which complex weakly with borate ions; and they will strongly resist elution from the column. Thus, there should be a relationship between the M , values of sugars (as determined by zone electrophoresis) and their affinity for a borate anion-exchanger: namely, the higher the M , value of a sugar, the more difficult it should be to elute that sugar from the borate anion-exchanger. The following sequences show the relative ease of elution96 and the M , values (in parentheses) for a series of simple sugars: ribose (0.77) >> fructose (0.90) > galactose (0.93) > glucose (1.00); and ribose (0.77) >> arabinose (0.96) > xylose (1.00). The correlation between M , value and affinity for the column is clear. Many separations of carbohydrates on borate anion-exchangers have been described by Khym, Zill, 9 6 * 97 These include the separation of pentoses, hexand their oses, heptoses, di- and tri-saccharides, deoxy sugars, sugar alcohols, and sugar phosphates. Other workerss8have studied sugar phosphates and methylated sugars,99and, in the majority of cases where a comparison may be made, D. H . Northcote, Biochem. J . (London), 68, 353 (1954). J. X . Khym and L. P. Zill, J . Am. Chem. SOC., 73,2399 (1951) ;74,2090 (1952). W. E. Cohn, in Ref. 76, p. 235. G. R. Noggle and L. P. Zill, Arch. Biochem. and Biophys., 41,21 (1952) ;M. A Chambers, L. P. Zill and G. R . Noggle, J . Am. Pharm. Assoc., 41, 691 (1952); J. X. Khym and W. E. Cohn, J . A m . Chem. SOC.,76, 1153 (1953); J. X. Khym, D. G. Doherty and W. E. Cohn, ibid., 76,5523 (1954) ;J. X. Khym and W. E. Cohn, Federation PTOC., 13, 241 (1954). (98) J. 0. Lampen, J . Biol. Chem., 204,999 (1953); M. Goodman, A. A. Benson and M. Calvin, J . A m . Chem. SOC.,77, 4257 (1955). (99) M. V. Lock and G. N. Richards, J . Chem. SOC.,3024 (1955). (94) (95) (96) (97)

ZONE ELECTROPHORESIS O F CARBOHYDRATES

115

the above noted relationship between the M , value and the column affinity obtains. An obvious value which stems from this correlation is that information may be provided (by 41, values) which will be of use in translating a microscale, zone-electrophoretic separation to a macroscale, borate anionexchange separation. It is of interest to note that borate-complex formation increases the affinity of a carbohydrate for a borate anion-exchanger and decreases its affinity for a carbon-Celite column.68