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Chromatographic Separation of Milk Proteins: A Review'

MAKOTO YAGUCHI and DYSON ROSE Division of Biology, National Research Council of Canada Ottawa, Ontario Abstract Ion Exchange Chromatography

The application of ion exchange chromatography and gel filtration to total skimmilk protein, casein, whey proteins, and enzymes are briefly reviewed.

During the past ten years highly discriminating chromatographic techniques have been adapted to and extensively used in studies of milk proteins. Ion exchange chromatography and gel filtration have made possible the isolation and purification of many previously unrecognized milk proteins, and must now be considered essential research techniques. Extensive reviews of work on milk proteins have been published by McI~enzie (132, 133) and by Rose et al. (199) and the reader is referred to these for information regarding the complexities of the milk protein system, and for characteristics of the individual proteins. The present paper deals only with the application of column chromatographic techniques. In the preparation of this paper, over 350 publications were reviewed. About 200 of these were selected (Tables 1 and 2) as containing technical information likely to assist a reader in adapting chromatographic methods to his own needs, and a smaller number were selected for technical discussion as examples of the utility of these methods. These selections were somewhat arbitra~T, no attempt was made to select the earliest publication of a technique, nor to include all variations of a technique. We offer our apologies to authors whose valuable contributions have been omitted. Most of the chromatographic media for the separation of milk proteins are readily divided into two classes; ion exchange media which separate proteins in relation to a net charge effect, and gel filtration media which separate proteins on the basis o size. These two types will be discussed separately, and each wilt be considered relative to total skimmilk proteins, caseins, whey proteins, and milk enzymes. 1 Issued as NRCC 12272.

Chromatography of proteins on ion exchangers involves the establishment o multiple electrostatic bonds between ionized groups on the surface of the exchanger and opposite charges on the proteins, followed by a selective release of these bonds by changes in the concentration or p i t of the eluant. Readers are referred to Sober and Peterson (213, 214), Peterson and Sober (179, 181) and Knight (1!6) for discussion of the principles and methodology. Synthetic exchange resins such as the Dowex and Amberlite products are extremely useful for amino acids and peptides and are sometimes useful for small proteins, but they tend to bind proteins strongly and thus to require rather severe conditions for elution. The ion exchange celluloses developed by Sober and Peterson in 1954 (178, 212), on the other hand, have high absorptive capacities yet bind proteins loosely. Both the anionic and cationic forms have therefore become important methods for the study of proteins and enzymes. More recently, ion exchange cross-linked dextrans (ion exchange Sephadex) and polyacrylamide gels (Bio-Gel CM and DM) hsve become available. Skimmilk proteins. I f caseins are solubilized by removal of most of the calcium (e.g. by prolonged dialysis at p H 7.0, 5 C) total skimmilk proteins can be partially fractionated by ion exchange chromatography. Association of caseins is temperature dependent at 0 to 35 C and casein aggregates bind more firmly to the exchanger than the disperse proteins. Consequently, at 25 C whey proteins elute ahead of all casein fractions (219, 220, 243), while at 5 C the pattern reproduced in Figure 1 is obtained (242). Under conditions of this test, both the K-casein (Peaks i to l) and the as-casein (peaks m to ~p) were contaminated with other caseins, suggesting that dissociation was n o t complete. However, these results indicate that partial fractionation can be achieved under conditions that are sufficiently mild to preserve a labile lipase activity that was associated, under these conditions, with the K-casein rich fraction. About 10 to 15% of the applied protein was not eluted with NaC1, but eluted with 0.2 s NaOtt. This






I 6.0








Fzo. 1. Elution pattern of dialyzed skimmilk (150 ml, 4.86 g protein) from DEAF,-ccllulose column (2.8 by 80 cm) at 5 C (Reference 242). The major component(s) of the identified peaks are: Peak a, 5,-casein and immunoglobulins; e, a-]actalbumin; f to h, fl-casein; i, fl-lactoglobulin; j to 1, ~-cascin;
m to p, a~-cascin.

fraction contained mixed caseins, which presumably had been absorbed as large aggregates. Caseins. The remarkable complexity (132, 199) of the casein system hindered the separation and purification of individual caseins, with the result that relatively little progress had been made before chromatographic methods became available. Use of these methods during the past 10 years has vastly expanded our knowledge of bovine caseins, and their use is being extended to the caseins of other species (Table 1). Because of the strong tendency for caseins to aggregate, ion exchange chromatography is more effective in the presence of a dissociating agent. Urea has been widely used for this purpose but its use presents several problems (94, 115, 127). At neutral or alkaline pH, cyanate accumulates in urea solutions and can give rise to artifacts by carbamylation of lysine and SH groups. Use of freshly prepared urea and low temperatures reduees cyanate accumulation (128), and cyanate can be removed with ion exchange resins (223). Waugh et al. (233) acidified urea solution to p H 3.5 for storage and adjusted to p H 7 just

before use. Methy]amine can also be used to "scavenge" cyanate (87). Dimethylformamide (DMF) can be used in lieu of urea, but its vapor is toxic so it should be used only in well ventilated rooms or hoods. Elution of protein from anion exchangers is usually achieved by increasing the concentration of chloride or phosphate. However, four types of eluant systems have contributed to the usefulness of ion exchange chromatography for caseins and these will be discussed briefly with examples of their use. a) Buffer, no dissociating and no reducing agents (68, 85, 86, 90, 98, 206). Groves et al. (85, 86) used a DEAE-eellulose colmnn and this type of eluant for the isolation of TScasein, T-casein, and fl-casein at p H 8.3 from either the fraction of casein soluble at p H 4.0 at 2 C, or from whole casein. TS-easein was not absorbed in the presence of 0.005 phosphate, T-casein eluted with 0.02 ~, and flcasein with 0.10 ~ phosphate. b) Buffer plus urea, no reducing agent (196, 197, 201, 225, 228, 233). Ribadeau-Dumas (196) and Ribadcau-Dumas et al. (197) chromatographed whole casein on a D E A E -




TABLE 1. Ion exchange chromatography of milk proteins. (Continued) Protein Skimmilk proteins, bovine Medium (dissociating agents) DEAE-cellulose rEAE-cellulose Caseins, bovine Whole casein DEAE-cellulose DEAE-cellulose (urea) DEAE-cellulose (urea, ME) DEAE-cellulose (DMF) SE-Sephadex Dowex 50WX4 DEAE-cellulose (urea) DEAE-cellulose (urea, ME) SE-Sephadex (urea) DEAE-cellulose DEAE-cellulose (urea) DEAE-eellulose (urea, ME) Phospho-cellulose (urea) DEAE-cellulose DEAE-cellulose (urea) TEAE-cellulose DEAE-eellulose DEAE-cellulose (urea) DEAE-cetlulose (urea, ME) DEAE-cellulose (DMF) CM-cellulose (urea, ME) CM-cellulose (DMF, ME) CM-Sephadex (urea, ME) DEAE-cellulose DEAE-Sephadex A-25 Dowex 50 2 References 114, 170, 219, 226, 242, 243 98

(bitter peptides) as-Caseins

60, 137, 206, 219 220, 245 196, 197, 200 136, 222, 230 6O 10 130
173, 224, 233, 234 52, 230 10 85, 86 66, 67, 68, 95, 96, 169, 225, 234 230 118 85, 86 228 99 4, 90, 92, 119, 137 173, 201~ 229~ 237 42, 136, 194, 195, 205~ 230 124 115 115 238 4, 119 43~ 126, 227 54


~, TS-Caseins K-Caseins

K-Casein maeropeptides

Caseins, other species Caseins, human

Caseins, swine K-Caseins, sheep Whey proteins, bovine Whole whey proteins

DEAE-eelIulose 154, 156, 158 DEAE-eellulose (urea or DMF) 3 DEAE-cellulose (urea, ME) 171, 172 DEAE-Sephadex A-25 126 DEAE-cellulose (urea) 239 DEAE-cellulose (urea, ME) 2 DEAE-cellulose AG 11A8 DEAE-cellulose DEAE-cellulose P-cellulose DEAE-cellulose CM-cellulose DEAE-cellulose P-cellulose 34, 81, 207, 219, 243 162 113, 215 16, 27, 80, 95, 215 81 97, 106, 113, 185 113 22, 80 80, 81

"Lactatbumin" fraction a-Lactalbumin

fl-Lactoglobulin Transferrin



TABLE 1. Ion exchange chromatography of milk proteins. (Concluded) Protein Lactoferrin Medium (dissociating agents) DEAE-cellulose Amberlite CG-50 Amberlite IRC-50 DEAE-cellulose DEAE-cellulose DEAE-cellulose, CM-cellulose DEAE-cellulose P-cellulose DEAE-Sephadex A-50 References 71, 217 174 72 83 58 16, 17 30, 34, 84, 125 84 112, 151, 152

Lactolliu Folate-binding protein Phosphoglycoprotein-a Immunoglobulins

Whey proteins, other species Whole whey proteins, Swine Transferrin, human Lactoferrin, human fleA-Globulin, human Laetoferrin, goat a-Laetalbumin, Guinea pig fLLaetoglobulin, goat fl-Laetoglobulin, swine Immunoglobulin, swine Enzymes, bovine Acid phosphatase Alkaline phosphatase Amino acid acyJamidase Lactoperoxidase

DEAE-celluloso DEAE-celluloso DEAE-cellulose CM-Sephadex C-50 DEAE-cellulose Amberlite CG-50 CM-cellulose DEAE-cellulose DEAE-eellulose DEAE-eellulose Amberlite IRC-50 DEAE-cellulose DEAE-cellulose Bio-Rex 70 Amberlite IRC-50 DEAE-Sephadex DEAE-cellulose, P-cellulose P-cellulose, DEAE-cellulose DEAE-cellulose DEAE-cellulose (DMF) Amberlite IRC-50 Amberlito IRC-50 DEAE-cellulose, P-cellulose

111 22, 31, 236 ]01, 129 101, 102, 129 155 174 26 107 105, 111 25 20 120 134 198 5, 149, 150 32, 33 80 51 59, 60, 63, 202, 242 60 35 19, 21 81

Lactose synthetase A protein Lipases Lysozyme (muramidase) Ribonuclease Enzymes, other species Lactoperoxidase, Sheep and goat Lactose synthetase A protein, human Lysozyme, human Ribonuelease, human

Amberlite IRC-50 DEAE-cellulose Amberlito IRC-50 C~-cellulose Amberlite MB-1, IRC-50

6 9 103, 104, 175 103 39

Abbreviations are: ME, mercaptoethanol; SDS, sodium dodecylsulfate; DMF, dimethylformamide ; DEAE, diethylamino-ethyl ; TEAE, triethylaminoethyl; CM, carboxymethyl.
J . ])AIRY SCIENCE ~?OL. 54, NO. 12

PROTEIN SEPARATION cellulose column with urea as a dissociating agent, and a sodium chloride elution gradient. Alpha s- and fl-casein were obtained in a moderately pure form with K-casein as the major contaminant. K-Casein eluted in two main peaks but also spread through many other fractions. Rose and Marier (201) attempted to p u r i f y ~-casein preparations with this system and considered the results unsatisfactory. This eluant system has also been used with reduced and alkylated casein smnples. Alkylation blocks the sulfhydryl groups and thus, for chromatographic purposes, is comparable to reduction of the SS groups and the separations achieved are similar to those discussed below. Reduced and alkylated K-casein components were fractionated by the urea-containing buffers by Woychik et al. (237) and Rose et al. (200) achieved quantitative estimation of the major caseins with this system; a typical chromatogram is presented in Figure 2. c) Buffer plus urea and mercaptoethanol (ME) (136, 194, 205, 222, 230). Thompson (222) added ME to the eluant and observed that ~-easein in its reduced form separated from fl- and %-casein. Mercier et al. (136)


used this eluant system on a preparative scale (10 g of casein) and several workers (136, 194, 195, 205) have used it for the fraetionation of K-casein components. d) Buffer, dimethylformamide (DMP) (115, 124). Mackinley and Wake (124) used D M F as dissociating agent in a fractionation of alky]ated K-casein. This system has also been used for isolation of para-K-casein (115). Under some conditions, cation exchangers give good separation of caseins (10, 115, 118, 238). SE-Sephadex has been used with the urea-ME eluant system, p i t 4.0, for fractionation of as-casein complex (10). P-cellulose has been used with ~rea, p i t 5.0, for purification of individual fl-easeins (118). These systems are of possible importance because the acid conditions prevent cyanate accumulation in urea solutions. Several workers have reported chromatographic separation of human caseins (3, 126, 154, ]56, 171, 172). Thus Nagasawa et al. (154, 158) used DEAE-cellulose chromatography at 10 C with buffers containing no urea and separated human casein into at least nine fractions, but no calcium sensitive fraction

Reference s0rnple

0 cO (kl .5





Or) Z LIJ O ..J


>: __


0 I-13_ 0















FIG. 2. Elution pattern for a 250 mg sample of reduced and a]kylated acid casein from DEAEcellulose column with NaC1 gradient in buffer containing 6.6 ~ urea (Reference 200). Fraction 1, TS-casein, 7-casein and para-K-Casein-like material; 2, z-casein, some 5,-casein; 3, fl-caseln; 4, a~-casein, some faster-moving components. J'. DAI~Y SOIENCE VOL. 54. NO. 12




similar to bovine as-casein was found, lqishikawa and Saito (171) also reported the absence of as-like fraction under these conditions. Malpress and Seid-Akhavan (126), on the other hand, used the cation exchanger, CM-Sephadex C-50, with urea, and obtained fractions corresponding to bovine a s- and to K-casein. Alais and Joll~s (3) obtained an as-casein and two K-casein like fractions from human milk; they used DEAE-cellulose and either 3.3 ~ urea, or 20% DMF with a sodium chloride gradient. When ME was included in the eluant, Nishikawa et al. (172) obtained eight fractions from human milk casein; these were reported to be homogeneous by polyaerylamide gel electrophoresis, and six fractions contained sialic acid and other carbohydrates. Recently, lqagasawa et al. (156) isolated electrophoretically homogeneous human fl-casein B after Sephadex G-150 gel filtration and DEAE-cellulose chromatography. The amino acid composition of human fl-casein B resembled that of cow fl-casein. Ion exchange chromatography has also been used to isolate K-casein:like fractions from sheep casein (2) and of as-, fl- and K-casein-like fractions from sow casein (239). Whey proteins. The major proteins of bovine whey have been relatively amenable to separation and purification by chemical and physical methods, and several had been thoroughly studied before chromatography came into common usage. Nevertheless, both ion exchange chromatography and gel filtration have been useful in this field, particularly for minor whey protein components, and proteins from other species, as they have provided milder and simpler separation techniques. Dissociating agents such as urea are not required for whey proteins, and may in fact be disadvantageous (1, 140). Schober et al. (207) separated whey proteins into eight fractions on DEAE-cellulose with step-wise changes in pH and sodium chloride concentration, and Yaguchi et al. (243) achieved an improved separation of alactalbumin and fl-lactoglobulin with step-wise increases in sodium chloride concentration at pH 7.0. The "albumin" fraction (soluble in half saturated ammonium sulphate) of whey protein has been separated into 10 fractions on DEAE cellulose by Szuchet-Derechin and Johnson (215, 216, 217, 218). Major fractions were filactoglobulin, a-lactalbumin, and bovine serum albumin. One minor fraction was identified as the red protein isolated from casein by Groves (79) by DEAE cellulose chromatography, and another as a blood serum transferrin; both of

these proteins contain iron. Groves (83) used a DEAE-cellulose column to isolate a nonironcontaining minor component which he named "lactollin" (Mol wt ca 43,000) and Ford et al. (58) used DEAE-cellulose followed by gel filtration to purify a folate-binding protein (Mol wt ca 35,000). DEAE-cellulose columns have also been used to separate genetic variants of fl-laetogIobulin (106, 113, 185). CM-cellulose has also been used for fractionation of the "albumin" fraction, the proteins were adsorbed at pH 4.6 and eluted by increasing pH (113). The "globulin" fraction of bovine whey proreins (insoluble in half-saturated ammonium sulphate) are largely immunoglobulins (30). The immunoglobulins are highly heterogeneous proteins that are very difficult to separate into individual components. However, partial separation into sub-groups of immunoglobulins has been possible by DEAE-cellulose (34, 84, 125) and DEAE-Sephadex (112, 151, 152) column chromatography. Milk enzymes. Milk contains a variety of enzymes (45, 209) that are distributed among the various classical milk protein fractions. The chromatographic procedures required for purification of these enzymes are thus essentially those used for the particular protein fraction of interest. However, successful isolation of enzymes requires preservation of enzymatic activity throughout the preparation. Both ion exchange chromatography and gel filtration can be performed under very mild conditions and are suitable for the isolation of many enzymes, provided the chromatographic conditions such as pH, temperature, kind and concentration of buffer, and dissociating agents, if necessary, are carefully selected to preserve the enzymatic activity. Acid phosphatase, lysozyme, and ribonuclease are basic proteins in milk whey. Amberlite IRC-50 resin was used for specific adsorption of these enzymes from the whey or directly from skimmilk, and found to be very effective in the initial purification of these enzymes (19, 20, 21, 35, 103, 104, 175). The acid phosphatase was further purified by column chromatography with Amberlite IRC-50 (21). Joll~s and Joll~s used CM-cellulose (103) and Amberlite IRC-50 (104) for further purification of human lysozyme, whereas Chandan et al. (35) and Parry et al. (175) preferred gel filtration with Sephadex G-50 in their final purification of human and bovine lysozymes. Bingham and Zittle (20) used an Amberlite IRC-50 column to isolate ribonuelease A, and Bingham and Kalan (19) separated ribonuclease A and



ribonuclease B on the same resin. During the isolation of lactoferrin and lactoperoxidase with DEAE cellulose and phosphaeellulose (80), Groves (81) obtained ribonuclease as a byproduct and achieved its crystallization. Various chromatographic methods have been used for the isolation of lactoperoxidase, an iron-containing enzyme in milk whey. Polis and Shmukler (186) used displacement chromatography with tricalcium phosphate and silicacelite columns in 1953. Morrison et al. (149) used Amberlite IRC-50 ion exchange chromatography and Morrison and Hultquist (150) used gel filtration with Sephadex G-100 to remove traces of two extraneous proteins. Rombauts et al. (198) used ion exchange chromatography with Bio-Rex 70, earboxylic acid resin, and reversed "salting-out" chromatography. More recently, Carlstrom (32, 33) succeeded in separating lactoperoxidase subfraetions by DE&E-Sephadex chromatography. Lipase activity of bovine milk is associated with the casein fraction. DEAE-cellulose was used by various workers (51, 60, 202, 242) to study its distribution and isolation. Since acid precipitated casein is devoid of the lipase activity, casein precipitated by ammonium sulfate (60) and by ultracentrifugation (202) or an extract from rennet-casein (59, 62) was used as starting material for the chromatographic separation. Dimethylformamide (DMF) was found to be an effective dissociating agent for the separation of lipase from a x-casein-rich fraction (59, 60). Protease (247) and amannosidase (135) are also associated with the casein fraction.
Gel Filtraflon

Gel filtration, which has also been called gel chromatography, exclusion chromatography, molecular sieve chromatography, and gel permeation chromatography, is a technique in which separation depends primarily on molecular size. Large molecules are completely excluded from the porous gel grains and emerge from the chromatographic bed at void volume. Molecules within a specific size range penetrate some gel pores and emerge later, followed by small molecules and ions that freely penetrate the gel pores. Suitable cross-linked gels were developed in 1959 by Porath and Flodin (189), and it soon became apparent that gel filtration would be an efficient method for the separation and isolation of proteins (57, 188) and for testing homogeneity and estimating molecular size (7, 8, 40, 235). The techniques are relatively simple, dilute buffer solutions serve as eluants,

separation occurs under very mild conditions, and the gels require no regeneration. Detailed discussions of the procedures have been presented by Determann (44) and by Fisher (55). Sephadex is the trade name for a series of cross-linked dextran gels which differ in the degree of cross-linking and hence in swelling properties and pore size. Generally speaking, Sephadex G-10, G-15, G-25 and G-50 are suitable for desalting (56, 183, 244) and fractionation of small peptides (12, 43, 54, 122, 130, 184), G-50 and G-75 for large peptides or small proteins (19, 35, 190) and G-100, G-150 and G-200 for proteins. Bio-gel is the trade name for a similar series (11 types, P-2 to P-300) (55) of cross-linked polyaerylamide gels. Agarose gels have fractionation ranges for considerably larger molecules or aggregates (50, 143, 153). Skimmilk proteins. Gel filtration of skimmilk had been attempted with Sephadex G-75 (146), G-100 (90, 146), G-150 (24) and G-200 (241) (Table 2). G-75 did not give a satisfactory separation (146). G-100 yielded three prorein fractions: casein complex, fl-laetoglobulin and a-lactalbumin. The effluent from G-200 (241) was separated into six fractions (Fig. 3), the first three of which contained casein. Fraction A contained ~-, as-, and fl-easein, probably as a complex; fractions B and C both contained a s- and fl-casein, but the proportion of %casein was considerably higher in B than in C. The last three fractions, D, E and F, were fllactoglobulin, a-laetalbumin and the dialysable non-protein components, respectively. When skimmilk is subjected to gel filtration with a dilute buffer as eluant the gel acts as an effective dialyser and removes inorganic calcium and phosphate from the micelles. This presumably leads to micellar disintegration and permits subsequent partial fractionation of the proteins. Morr and Josephson (143) and Murphy et al. (153) have successfully separated size-ranges of intact micelles on agarose (Sepharose 2B and 4B) with complex buffer mixtures designed to minimize the dialysis effect. Boulet et al. (24) examined the mineral composition of the casein micelle obtained with Sephadex G-150. The gel filtration technique is also used to separate milk protein from the low molecular weight components such as lactose, salts, riboflavin, amino acids, and other soluble materials. The application of this technique for recovery of proteins from skimmilk and cheese whey (142, 162, 168, 183), production of lactose-free milk (41) and protein enriched milk (203), and analysis of soluble calcium,
J. DAffY S C ~ C E ~r0L. 54, NO. 12




E 1.5

O q
I(~ I.C

7 <~ m nO 0.~
O0 m








Fie. 3. Gel filtration of skinlmilk (3 m]) on a 2.5- by 80-cm column of Sephadex G-200 at 4 C (Reference 241). Eluant: 0.02 ~ Na-phosphate, pH 7.0. Fraction A, ~-casein, a~-casein, fl-casein; B, a~-casein ~ fl-casein; C, fl-casein > a~-casein; D, fl-lactoglobulin; E, a-lactalbumin; F, dialyzable materials. phosphate and citrate in nfilk (244) have been reported. Caseins. Although casein micelles are complex and very large, monmneric caseins are of relatively low molecular weight (20 to 30,000) and can penetrate the gel pores of Sephadex G-200, G-150, and G-100. To achieve separation on these gels it is necessary to dissociate the casein aggregates, and the separation achieved is primarily dependent on the degree of dissociation rather than on the molecular weights of the nlonomeric forms. Urea, dimethylformamide, sodium dodocyl sulphate (SDS), guanidium chloride, extremes of p H and low temperatures, with and without reducing agents such as 2-mercaptoethanol (ME) and dithiothreitol (DTT) have been used to induce the desired type and degree of dissociation. I n the presence of both reducing and dissociating agents, all three major caseins dissociate extensively, and effective separation is not achieved on Sephadex G-200 (37). I n the absence of reducing agents, K-casein does not dissociate to the same extent as a s- and flcasein, so elutes at the void volume; highly purified ~-casein has been obtained in the presence of urea at p H 8.6 (240), or with SDS and EDTA (37). I n an interesting adaptation of gel filtration, Kenkare and Hansen (85) have studied the reversible temperature-induced dissociation of whole a-casein on G-100 Sephadex in a jackJ. DAIRY SCIENCE OL. 54, NO. 12

eted, thermostated column. At room temperature, a s- and K-casein eluted as a single peak at void volume; at 54 C and above as-casein was retarded and only K-casein eluted at the void volume. Gel filtration has also been used to study the effect of various treatments. Thus, Nakanishi and Ito (163, 164) studied the effect of heating and of frozen storage of milk on the properties of whole casein; both heating and frozen storage increased the proportion of casein eluted at the void volume. Nakai et al. (159, 160, 161) examined the gel filtration properties of chemically modified K-casein and paraK-casein, and estimated the monomeric molecular weight of K-casein as 20,500. Downey et al. (50) studied the dissociation of fl-casein from casein micelles; at 5 C about 65% of the flcasein could be removed without apparent micelle disintegration. Nagasawa et al. (156) used gel filtration as a preliminary step in the preparation of fi-casein in K-casein from human milk. Whey proteins. Whey proteins differ markedly in molecular weights, have less tendency to aggregate than the caseins, can be separated by gel filtration in the absence of dissociating agents. As with ion exchange chronmtography, use of such agents may, in fact, be detrimental (1, 140). I n the presence of both dissociating and reducing agents, fl-laetoglobulin was eluted as subunits which have a molecular weight of 18,600 (40).




TABLE 2. Gel filtration of milk proteins. (Continued) Protein Skimmilk proteins, bovine Medium (dissociating agents) Sepharose 2B Sepharose 4B Sephadex G-200 Sephadex G-150 Sephadex G-100 Sephadex G-75 Sephadex G-50 Sephadex G-25 Caseins, bovine Whole casein Sepharose Sephadex G-200 Sephadex G-200 (SDS) Sephadex G-150 (urea) Sephadex G-50 (urea) Sephadex G-50 Sepharose 2B Sephadex G-200 Sephadex G-1OO Sephadex G-200 Sephadex G-200 (SDS) Sephadex G-200 (SDS, DTT) Sephadex G-200 Sephadex G-2O0 (NaOIt) Sephadex G-150 (urea) Sephadex G-100 Sephadex G-100 Sephadex G-75, G-50 Sephadex G-25 Sephadcx G-50 Sephadex G-25 Sephadex G-15 Bio-Gel P-6 Bio-Gel P-10 Sephadex G-150 Sephadex Sephadex Sephadex Sephadex Sephadex Sephadex Sephadex G-200 G-100 G-50 G-25 G-10, Bio Gel P-2 G-100 G-75 References 49, 143 143 49, 139, 174, 241 24 91, 93, 121, 139, 141, 143, 146, 167 146 70, 142, 147 41, 70, 148, 183, 203 50, 143, 153 37, 50, 60, 153, 163, 241 37 240 241 210 77 176 109, 110 15 37 37 165 159, 160, 161 108, 240 166 100 14, 88 14, 88, 130, 184 210 12, 227 12, 43, 54, 119, 227 227 54 117 156 110, 123, 144, 167 144, 231 142 148 162 166 190, 191 27 27, 29 4O 204
J . DAIR: S O I ~ C E
YOL. 54, No. 12

as-Casein and K-casein as-Casein K-Casein

Temperature-sensitive Casein Casein peptides ~-Casein maeropeptides

Casein-associated glyeoproteins Caseins, other species Whole casein, human Whey proteins, bovine Whole whey proteins

"Lactalbumin" fraction

a-Lactalbumin (lactose synthetase B protein) B-Lactoglobulin

Sephadex G-100 Bio-Gel P-30 Bio-Gel A-SM (urea or guanidinm B1, and ME) Sephadex G-200



TABLE 2. Gel filtration of milk proteins. (Concluded) Protein fl-Lactoglobulin Lactoferrin Folate-binding protein ~-1, M-2, glycoproteins Proteose-peptone component 8 Immunoglobulins Immunoglobulins, glycopeptides Whey proteins, other species Whey proteins, Human Guinea pig Swine Folate-binding proteins, Human Lactoferrin, Human Goat Transferrin, Rabbit Immunoglobulins, Human Human Swine Rabbit Enzymes, bovine Alkaline phosphatase Lactoperoxidase Lactose synthetase A protein Lactose synthetase A and B proteins Lipases Medium (dissociating agents ) Sephadex Sephadex Sephadex Sephadex G-100 G-100 G-150, G-75 G-100, G-75 References 140, 145 174 58 16, 17, 18 117' 30, 75, 84, 152 84 61 125 158 26 111 58 102 174 13 11, 78, 89 78 25 208 65 38 81 51

Bio-Gel P-10 Sephadex G-200 Sephadex G-100 Bio-Gel P-300 Sephadex G-50, G-25 Sephadex G-150 Sephadex G-100 Sephadex G-100 Sephadex Sephadex Sephadex Sephadex Sephadex Sephadex Sephadex Sephadex G-75 G-150 G-100 G-200 G-200 G-100 G-200 G-200

Sagarose SAG-6 Sephadex G-200 Sephadex G-200 Sephadex G-200

Lysozyme (muramidase) Ribonuclease Enzymes, other species Lactose synthetase A protein, human Lactose synthetase A and B proteins, human, sheep, goat Lysozyme, human Ribonuclease, human a-Amylase, human Acid phosphatase, human

Bio-G~ P-30, Sephadex G-100 27, 28 Sepharose 2B 49 Sephadex G-200 46, 47, 48, 49, 59, 60, 232 Sephadex G-50 36, 232 Sephadex G-25 63, 64 Sephadex G-50 35 Sephadex G-75, G-25 19 Sephadex G-200 81

Sephadex G-50 Bio-Ge[ P-30 Sephadex G-100, G-75, G-25 Sephadex G-50 Sephadex G-50 Sephadex G-100 Sephadex G-200

9 27 104 103, 175 39 76 74

Abbreviations are:SDS, sodium dodecyl sulfate; DMF, dimethylformamide; ME, mercaptoethanol; DTT, dithiothreitol.



With Sephadex G-75 and triethylamine-acetic acid buffer, p H 4.6 Preaux and Lontie (123, 190, 191) obtained four peaks from a total whey protein preparation; the first peak (void volume) contained several proteins. With Sephadex G-100, this first peak separated into three components, so that the following six fractions were obtained: Peak 1 (void volume ) --residual casein and aggregates, 2--immunoglobulins, 3 - bovine serum albumin, 4--fi-lactoglobulins, 5 - minor components immunologically related to a-lactalbmnin, and 6--a-lactalbumin. Not all of these fractions were electrophoretically pure. I-Ieat-induced changes of whey proteins frequently result in formation of protein aggregates, and thus cause changes in gel filtration patterns (110, 123, 143, 144, 204). Sawyer (204) reported separation of heated fl-lactoglobulin on Sephadex G-200 and Kenkare et al. (110) examined the gel filtration properties of heated whey proteins on Sephadex G-100, and found that the amount of protein eluted in the void volume increased at the expense of the fllactoglobulin and a-lactalbumin peaks. Morr and Josephson (144) and Morr (141) found that heating aggregated whey proteins to intermediate sized particles which were excluded from Sephadex G-100 and G-200, and this aggregation was dependent on thioldisulfide interaction. Lonti and Preaux (123) reported changes in the gel filtration patterns of milk serum prepared from skimmilks which had been subjected to various commercial heat-treatments. Since most of the heat-induced complexes or aggregates are excluded from Sephadex gels, Agarose gels may be more suitable for fractionation of these complexes (143). Gel filtration has also been used for the isolation of minor whey proteins (Table 2), to check the purity of commercial preparations of whey proteins (145), to isolate glycopeptides of immunoglobulins (125), and to study the composition of whey proteins from other species. M i l k enzymes. Gel filtration is a very useful technique for milk enzymes because it separates them under very mild conditions and provides rapid desalting when eluted with water or dilute buffer. Also, the molecular size of the enzyme can be estimated even before purification is achieved. Various gels have been used to study the distribution and isolation of milk lipases. Chartdan and Shahani (36) isolated a lipase from clarifier slime using Sephadex G-5O, and found that the molecular weight was 7,000. Fox and Tarassuk (59) used Sephadex G-200 for the isolation of a lipase from skimmilk, and estimated its molecular weight to be about 210,000.

Gaffney et al. (62) also prepared lipases from skimmilk, and observed that most of them penetrated into Sephadex G-25 gels (63, 64). Dowhey and Andrews (46, 47, 48) obtained several milk ]ipase fractions which differed in molecular weights and substrate specificities. Downey and Murphy (49) studied the distribution of endogenous milk lipase activity, and association of pancreatic lipase with casein complexes, with Sepharose 2B and Sephadex G-200. The lipoprotein particles from the membrane surrounding the fat globules contain most of the alkaline phosphatase of cream, but an opalescent layer above the casein pellet obtained by centrifugation of the skimmilk also contains alkaline phosphatase. The alkaline phosphatase and phospholipid of the opalescent material were eluted from an Agarose SAG-6 colmnn in the void volume and were separated from contaminating casein (65). Gel filtration media have also been used successfully in the isolation of several other ei1zymes in bovine and human milk serum. Lactose synthetase A protein has a molecular weight larger than B protein (a-lactalbumin), and Bio-Gel P-30 was found to separate the two subunits completely into two fractions (27, 28). Bingham and Kalan (19) used a large Sephadex G-75 column (9 126 cm) to separate ribonuclease from the major portion of a whey protein fraction. Groves (81) used Sephadex G-200 to isolate ribonuclease from chromatographic fractions which also contained lacto-lactoperoxidase. Sephadex G-50 (35, 175) and Sephadex G-75 (104) were used to isolate milk lysozymes and Sephadex G-100 (104) was used to estimate the molecular weight of human milk lysozyme. Desalting of purified milk enzymes with Sephadex G-25 (19, 104), and Sephadex G-50 columns (39, 103) was rapid and complete, avoiding the loss of enzymatic activity that might occur during prolonged dialysis.
Concluding Comments During the past ten years, chromatographic methods have been applied extensively to isolate many individual components of the casein and albumin fractions of bovine milk, and these two chromatographic methods are probably more frequently used than any other fractionation procedures. Multiple purification with both cation and anion exchange chromatography and gel filtration are very effective in isolating and purifying minor components (e.g. 59, 150, 156). The chromatographic methods combined with conventional fractionation procedures may eventually enable isolation of all


YAGUCHI AND ROSE (15) Beveridge, H.J.T., and S. Nakai. 1970. Effects of chemical modification with 2p h e n y l - l . 4 - d i b r o m o a c e t o i n on asl-casein. J. Dairy Sci., 53:1532. (16) Bezkorovainy, A. 1965. Comparative study of the acid glycoproteins isolated from bovine serum, colostrum and milk whey. Arch. Biochem. Biophys., l l 0 : 558. (17) Bezkorovainy, A. 1967. Physical and chemical properties of bovine milk and colostrum whey 1~-1 gIycoproteins. 5. Dairy Sci., 50 : 1368. (18) Bezkorovalny, A., and D. Grohlich. 1969. Separation of the bovine colostrum M-1 glycoprotein into two components. Biochem. 5., 115 : 817. (19) Bingham, E. W., and E. B. Ka]an. 1967. Ribonuclease B of bovine milk. Arch. Biochem. Biophys., 121: 317. (20) Bingham, E. W., and C. A. Zittle. ]963. Purification and properties of acid phosphatase in bovine milk. Arch. Biochem. Biophys., 101: 471. (21) Bingham, E. W., and C. A. Zittle. ]964. Ribonuclease of bovine milk: Purification and properties. Arch. Biochem. Biophys., 106 : 235. (22) Blanc, B., and H. Isliker. 1961. Isolement et Caracterisation de la Proteine Rouge Siderophile du Lait m a t e r n e h La Lactotransferrine. Bull. Soc. Chlm. Biol., 43:929. (23) B]att, W. F., S. M. Robinson, 1~. M. Robbins, and C. A. Sarovis. 1967. An ultrafiltration membrane for the resolution and purification of bovine alpha-lactabumin. Anal. Biochem., 18: 81. (24) Boulet, ~ . , A. Yang, and R. R. Riel. 1970. Examination of the mineral composition of the mlcelle of milk by gel filtration. Canadian J. Biochem., 48: 816. (25) Bourne, F. J. 1969. I g A immunoglobulin from porcine milk. Biochim. Biophys. Acta, 181 : 485. (26) Brew, K., and P. 1~7. Campbell. 1967. The characterization of the whey proteins of guinea-pig milk. The isolation and properties of a-lactalbumln. Bioehem. J., 102 : 258. (27) Brodbeck, U., W. L. Denton, N. Tanahashl, and It[. E. Ebner. 1967. The isolation and identification of the B protein of lactose synthetase as a-lactalbumln. 5. Biol. Chem., 242: 1391. (28) Brodbeck, U., and K. E. Ebner. 1966. The subcellular distribution of the A and B proteins of lactose synthetase in bovine and mammary tissue. J. Biol. Chem., 241 : 5526. (29) Brodbeck, U., and K. E. Ebner. 1966. Resolution of a soluble lactose synthetase into two protein components and solubilizatlon of mlcrosomal lactose syntheCase. J. Biol. Chem., 241:762. (30) Butler, J. E. 1969. Bovine immunoglobulins: A review. 5. Dairy Sci., 52: 1895.

the i n d i v i d u a l c o m p o n e n t s of casein a n d alb u m i n fractions, b u t o t h e r relatively new techniques such as p r e p a r a t i v e gel electrophoresis, electrofocusing, column electrophoresis, memb r a n e ultrafiltration, c o u n t e r - c u r r e n t distribution, a n d zonal u l t r a c e n t i f u g a t i o n will also be useful f o r s e p a r a t i n g milk p r o t e i n s (23, 131, 177, 182, 221). References (1) Akroyd, P. 1965. Aerylamide-gel electrophoresis of fl-lactoglobulins stored in solutions at p H 8:7. Nature, 208: 488. (2) Alais, C., and P. Joll~s. 1967. Isolation, purification and analysis of two K-caseinlike fractions from sheep casein. J. Dairy Sci., 50: 1555. (3) Alais, C., and P. Jollbs. 1969. Chromatographic purification of human K-casein. J. Chromatography, 44: 573. (4) Alais, C., N. Kiger, and P. Jollbs. 1967. Action of heat on cow d-casein. Neat caseino-glycopeptide. J. Dairy Sci., 50 : 1738. (5) Allen, P. Z., and M. Morrison. 1963. Lactoperoxidase. IV. Immunological analysis of bovine lactoperoxidate preparations obtained by a simplified fractionation procedure. Arch. Biochem. Biophys., 102: 106. (6) Allen, P. Z., and M. Morrison. 1966. Lactoperoxidase. VI. Immunochemical studies on lactoperoxidate from the milk of several species. Arch. Biochem. Biophys., 113:540. (7) Andrews, P. 1964. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Bioehem. J., 91: 222. (8) Andrews, P. 1965. The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. 5 , 96: 595. (9) Andrews, P. 1969. Lactose synthetase A protein from human milk. Abstr. Biochem. 5 , 111: 14. (10) Annan, W. D., and W. Manson. 1969. Fractlonation of the alpha-casein complex of bovine milk. 5. Dairy Res., 39: 259. (11) Axelsson, H., B. G. 5ohansson, and L. Rymo. 1966. Isolation of immunoglobulln A ( I g A) from human colostrum. Acta Chem. Scand., 20: 2339. (12) Baker, B. E., and P. C. Hwang. 1967. Casein. V I I I . Isolation of a glycopeptide from an enzymic hydrolysate of d-casein. 5. Dairy Sci., 50: 1206. (13) Baker, E., D. C. Shaw, and E. It. Morgan. 1968. Isolation and characterisation of rabbit serum and milk transferrins. Evidence for difference in sialie acid content only. Biochemistry, 7 : 1371. (14) Bennich, H. 1961. Gel filtration of tryptic hydrolysates of a-casein. Biochim. Biophys. Acta, 51: 265.



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