RESEARCH

PAPERS

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.
Introduction

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

1725

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YAGUOHI AND ROSE

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I

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200

300

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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
,T. DAIRY SCIENCE V0L. 54, NO. 12

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 -

PROTEIN

SEPARATION

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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

]]-Caseins

~, TS-Caseins K-Caseins

para-K-Casein
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
J. DAIRY SCIEI~CE VOL. 54, NO. 12

"Lactatbumin" fraction a-Lactalbumin

fl-Lactoglobulin Transferrin

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YAGt~Cm AND ROSE

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)

1729

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

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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

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YAGUCHI A N D

ROSE

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
ft. DAIRY SCIENCE VOL. 54, NO. 12

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

PROTEIN SEPARATION

1731

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

1732

A G U C H I AND ROSE

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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).

PROTEIN

SEPARATION

1733

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

1734

YAeUOHI AND ROSE

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.
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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
J. ])AIRY SCIENCE VOL. 54, NO. 12

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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.
J. DAIRY SCIENCE VOL. 54, NO. 12

PROTEIN SEPARATION

1737

(31) Bnttkus, H., J. R. Clark, and R. E. Feeney. ]965. Chemical modification of anlino groups of transferrins: Ovotransferrin, human serum transferrin, and human lactotransferrin. Biochemistry, 4: 998. (32) CarlstrSm, A. 1969. Lactoperoxidase. Identification of multiple forms a n d their interrelationships. Acta Chem. Scand., 23: 171. (33) CarlstrSm, A. ]969. Physical and compositional investigations of the subfractions of lactoperoxidase. Acta Chem. Scand., 23 : 185. (34) Carroll, E. 3. 1961. Whey proteins of drying-off secretions, mastitie milk, and eolostrum separated by ion-exchange cellulose. J. Dairy Sei., 44: 2194. (35) Chandan, R. C., R. M. Parry, and K. M. Shahani. 1965. Purification and some properties of bovine milk lysozyme. Bioehim. Biophys. Acta, 110: 389. (36) Chandan, R. C., and K. M. Shahani. 1963. Purification and characterization of milk lipase. I. Purification. J. Dairy Sei., 46 : 275. (37) Cheeseman, G. C. 1968. A preliminary study by gel filtration and ultracentrifugation of the interaction of bovine milk caseins with detergents. 3. Dairy Res., 35 : 439. (38) Copius Peereboom, 3. W. 1968. Studies on alkaline milk phosphatase. II. Occurrence of various phosphatase isozymes in dairy products. Netherlands Milk Dairy ft., 22 : 137. (39) Dalay, B. K., 3. R. Vakil, and K. M. Shahani. 1968. Isolation of human milk ribonuclease. Abstr. J. Dairy Sci., 51:940. (40) Davison, P. F. 1968. Proteins in denaturing solvents: Gel exclusion studies. Science, 161: 906. (41) De Koning, P. 3. 1962. Gel filtration, a new method applied for the preparation of lactose-free milk. Netherlands Milk Dairy J., 16: 210. (42) De Koning, P. ft, Van Rooijen, P. 3-, and Kok, A. 1966. Location of amino acid differences in the genetic variants of K-casein A and B. Biochem. Biophys. Res. Commun. 24: 616. (43) Delfour, A., C. Alais, and P. Jolles. 1966. Structure of cow's r-caseinoglyeopeptide: the N-terminal octadeeapeptide. Chimia, 20: 148. (44) Determann, H. 1968. "Gel Chromatography". Springer-Verlag. New York Inc., New York City. (45) Dowben, R. M., J. R. Brunner, and D. E. Philpott. 1967. Studies on milk fat-globule membranes. Biochim. Biophys. Acta, 135: 1. (46) Downey, W. K., and P. Andrews. 1965. Gel filtration applied to the study of lipases and other esterases. Biochem. J., 94 : 642. (47) Downey, W. K., and P. Andrews. 1966.

(48) (49)

(50)

(51)

(52)

(53)

(54)

(55) (56) (57) (58) (59) (60)

(61) (62)

(63)

(64)

Studies on the properties of cow's milk tributyrinases and their interaction with milk proteins. Biochem. ft., 101: 651. Downey, W. K., and P. Andrews. 1969. Evidence for the presence of several lipases in cow's milk. Bioehem. J., 112:559. Downey, W. K., and R. F. Murphy. 1970. Association of lipases with miceller and soluble casein complexes. J. Dairy Res., 37 : 47. Downey, W. K., 1~. F. Murphy, and S. A. Aherne. 1969. Dissociation of fl-easein from casein micelles and complexes. Abstr. Biochem. ft., 115: 21. Ebner, K. E., N. Tanahashi, U. Brodbeck, and I. Kiyosawa. 1968. Purification and properties of the A protein of lactose synthetase. Abstr. Federation Proc., 27: 784. Farretl, H. M. Jr., M. P. Thompson, and B. Larsen. 1971. Verification of the occurrence of the asl-easein A allele in red Danish cattle. J. Dairy Sci., 54: 423. Feeney, R. E., and ~. G. Allison. 1969. Evolutionary Biochemistry of Proteins: Homologous Proteins from Avian Egg Whites, Blood Sera, Milk and Other Substances. Wiley-Interscience, New York City. Fiat, A. M., C. Alais and P. Jollbs. 1968. Contribution to the study of the linkage between the peptide and sugar moieties in cow's r-casein. Chimia, 22: 137. Fisher, L. 1969. An Introduction to Gel Chromatography. North-Holland Publishing Company. Amsterdam, The Netherlands. Flodin, P. 1961. Methodological aspects of gel filtration with special reference to resalting operations. 3". Chromatogr., 5: 103. Flodin, P. 1962. Dextran gels and their application in gel filtration. Thesis. University of Uppsala, Sweden. Ford, 5. E., D. N. Salter, and K. J. Scott. 1969. The folate-binding protein in milk. 3-. Dairy Res., 36: 435. Fox, P. 1% and N. P. Tarassuk. 1968. Bovine milk ]ipase. I. Isolation from skimmilk. 3-. Dairy Sci., 51: 826. Fox, P. F., M. Yaguchi, and N. P. Tarassuk. 1967. Distribution of lipase in milk proteins. II. Dissociation from r-casein with dimethylformamide. 3. Dairy Sci., 50: 307. Froese, A. 1971. Isolation of dinitrophenylspecific antibodies from bovine eolostrum. Canadian 5. Biochem., 49: 522. Gaffney, P. J. Jr., W. ft. Harper, and I. A. Gould. 1965. Kinetic properties of individual components of the lipase system of skimmilk. 3. Dairy Sci., 48: 767. Gaffney, P. J. Jr., W. J. Harper, and I. A. Gould. 1966. Distribution of lipase among components of a water extract of rennet casein. 3. Dairy Sci., 49: 921. Gaffney, P. 3. Jr., W. 3. Harper, and I. A. Gould. 1968. Characteristics of lipase-rich fractions of milk protein. 3. Dairy Sci.,
J . DAIRY SCIENCE ~-oL. 54, No. 12

1738

YAGucm AND ROSE ponents of casein and other phosphoproteins in milk. A review. J. Dairy Set., 52 : 1155. Groves, M. L., 5. J. Basch, and W. G. Gordon. 1963. Isolation, characterization and amino acid composition of a new crystalline protein, lacto]lin, from milk. Biochemistry, 2 : 814. Groves, M. L., and W. G. Gordon. 1967. Isolation of a new glycoprotein-a and a 5' G-globulin from individual cow milks. Biochemistry, 6: 2388. Groves, M. L., and W. G. Gordon. 1969. Evidence from amino acid analysis for a relationship in biosynthesis of */-caseins and fl-easeins. Biochim. Biophys. Acta, 194: 421. Groves, M. L., T. L. McMeekin, N. J. Hipp, and W. G. Gordon. 1962. Preparation of fl- and "y-caseins by column chromatography. Biochlm. Biophys. Acta, 57: 197. Hardy, S. 5. S., C. K. Xurland, P. Voynow, and G. Mora. 1969. The ribosomal proteins of Escherlchla colt. I. Purification of the 30S ribosomal proteins. Biochemistry, 8: 2897. Harwalkar, V. R., and 5. A. E]liott. 1971. Isolation of bitter and astringent fractions from Cheddar cheese. J. Dairy Sci., 54: 8. Havez, R., J. P. Muh, M. Bonte, and G. Biserte. 1966. Study of "/-A-globullns of human colostrum. Clinica Chim. Acta, 15: 7. Hill, R. D. 1963. The preparation of Kcasein. 5. Dairy Res., 30: 101. Hill, 1~. D. 1964. The eysteine content of casein micelles. J. Dairy Res, 31: 285. Hill, R. D., and R. R. Hansen. 1963. The effect of preparative conditions on the composition of the K-casein complex. 5. Dairy Res., 30: 375. Hill, R. D., and R. R. Hansen. 1964. The separation o milk proteins on dextran gel. 5. Dairy Res., 31: 291. Hill, R. J., and R. G. Wake. 1969. Further studies on the origin and nature of the bovine para-K-casein components. Biochim. Biophys. Acta, 175: 419. Hoagland, P. D. 1966. Acylated fl-caseins: Electrostatic interactions and aggregation. 5. Dairy Sci., 49: 783. Hoagland, P. D. 1968. Acylated fl-caseins: Effect of alkyl group size on calcium ion sensitivity and on aggregation. Biochemlstry, 7: 2542. Hunziker, H. G., and N. P. Tarassuk. 1965. Chromatographic evidence for heat-induced interaction on a-]actalbunfin and fl-lactoglobulin. 5. Dairy Sci., 48: 733. Igarashi, Y., and Z. Saito. 1967. Column chromatography of skim milk and buttermilk on triethylaminoethyl cellulose. Bull. Fac. Agr. Hirosaki Univ., 13:44. Igarashi, Y., and Z. Saito. 1968. Separation and some properties of temperature sensitive fraction in casein. Japanese J. Zootechnlcal Sci., 39: 78.

5] : 1161. (65) Gammack, D. B., and B. B. Gupta. 1967. The isolation of lipoprotein particles from bovine milk by a gel filtration procedure. Abstr. Biochem. J., 103: 72. (66) Garnier, J., B. Ribadeau-Dumas, and G. Mocquot. 1964. A new method for the preparation of an immunologlcally homogeneous fl-casein. J. Dairy Res., 31: 131. (67) Gehrke, C. W., 1) . C h u n , and Y. H. Oh. 1966. Isolation and polyacrylamide gelurea electrophoretie characterization of flcasein. Separation Sci., 1: 431. (68) Gehrke, C. W., C. W. Freeark, Y. ~ . Oh, and P. W. Chum 1964. Isolation of eleetrophoretically pure fl-caseins. Anal. Biochem., 9 : 423. (69) Ge]otte, B. 1960. Studies on gel filtration: Sorption properties of the bed material Sephadex. J. Chromatogr., 3:330. (70) George, W. H. S. 1962. Separation of strontium from milk and protein solutions by gel filtration. Nature, ]95: 155. (71) Gordon, W. G., M. L. Groves, and 5. 5. Basch. 1963. Bovine milk "red protein": Amino acid composition and comparison with blood transferrin. Biochemistry, 2: 817. (72) Gordon, W. G., M. L. Groves, and J. 5. Basch. 1963. Isolation of an iron-binding protein from cow's milk. Biochim. Biophys. Acta, 60: 410. (73) Got, R. 1965. Fractionation of human ]actoserum proteins. Clinica Chim. Acta, 11:432. (74) Got, R., 5. Ferez, and Y. Goussault. 1966. Presence of an acid phosphatase of the prostatic type in human colostrum. Clinica Chim. Acta, 14: 842. (75) Gough, P., R. Jenness, and R. K. Anderson. 1966. Characterization of bovine immunoglobulins. Abstr. J. Dairy Set., 49: 718. (76) Goussault, Y., R. Got, and A. Marnay. 1967. Preparation of a-amylase from human colostrum by Sephadex filtration. Protides Biol. Fluids, 14: 621. (77) Green, M. L. 1971. The specificity for Kcasein as the stabilizer of a~-casein and fl-casein. I. Replacement of K-casein by other proteins. 5. Dairy ICes., 38: 9. (78) Grey, H. M., C. A. Abel, W. 5. Yount, and H. G. Kunkel. 1968. A subclass of human -y A-globulin (TA2) which lacks the disulfide bonds linking heavy and light chains. J. Exp. Med. 128: 1223. (79) Groves, IV[. L. 1960. The isolation of a red protein from milk. J. Amer. Chem. Soc., 82 : 3345. (80) Groves, M. L. 1965. Preparation of some iron-binding proteins and a-lactalbumin from bovine milk. Biochim. Biophys. Acta, 100 : 154. (81) Groves, M. L. 1966. Crystalline ribonuclease from bovine milk. 5. Dairy Sci., 49 : 204. (82) Groves, )~. L. 1969. Some minor coral. DAIRY SCIENCE V0L. 54, NO. 12

(83)

(84)

(85)

(86)

(87)

(88)

(89)

(90) (91) (92)

(93)

(94)

(95)

(96)

(97)

(98)

(99)

PtCOTEIN SEPARATION Igarashi, Y., and Z. Saito. ]970. Some properties of temperature-sensitive casein in cow's milk. Japanese J. Zootechnical Sei., 41 : 262. (1Ol) Johansson, B. 1960. Isolation of an ironcontaining red protein from human milk. Aeta Chem. Scand., 14: 510. (102) Johansson, ]3. 1969. Isolation of crystalline lactoferrin from human milk. Acta Chem. Scand., 23: 683. (103) Joll~s, P., and J. Joll~s. 1961. Lysozyme from human milk. Nature, 192: 1187. (104) Joll~s, J., and P. Joll~s. 1967. Human tear and human milk lysozymes. Biochemistry, 6 : 411. (105) Jones, S. ]3., and E. ]3. Kalan. 1971. Modified procedure for isolation of a major swine whey protein. J. Dairy Sci., 54: 288. (106) Ka]an, E. ]3., R. Greenberg, M. Walter, and W. G. Gordon. 1964. Chemical properties of fl-iactoglobulin A, ]3, and C. ]3iochem. ]3iophys. Res. Comm., 16: 199. (107) Kalan, E. ]3., A. J. Neistadt, and P. D. ttoagland. 1967. Heterogeneity of goat fllactoglobuHn. Abstr. J. Dairy Sci., 50: 949. (108) Kanamori, M., Miyoshl, M., and Ibyki, F. 1969. K a p p a casein of bovine milk. Purity of kappa caseins prepared by several different methods. Eiyo To Shokuryo, 22: 639. (109) Kenkare, D. ]3., and P. M. T. Hansen. 1967. Reversible heat-induced dissociation of alpha-casein complex. J. Dairy Sci., 50: 135. (110) Kenkare, D. ]3, C. V. Morr, and I. A. Gould. 1964. Factors affecting the heat aggregation of proteins in selected skimmilk sera. J. Dairy Sci., 47: 947. (Ill) Kessler, E., and K. Brew. 1970. Whey proteins of pig's milk. Isolation and characterization of a fl-lactoglobulin. Riochim. ]3iophys. Acta, 200: 449. (112) KickhSfen, ]3., D. K. Hammer, and S. Scheel. 1968. Isolation and characteristics of "I-G type immunoglobu]ins from bovine serum and eolostrum, tIoppe-Seyler's Z. Phys. Chem., 349: 1755. (113) Kiddy, C. A., R. E. Townend, W. W. Thatcher, and S. N. Timasheff. 1965. flLactoglobulin variation in milk from individual cows. J. Dairy Res., 32: 209. (114) Kim, Y. K., S. Arima, and Y. Hashimoto. 1967. Sialie acid in milk. I I I . Sialic acld in normal milk and in the fractions of milk proteins obtained by diethylaminoethylcellulose column. J. Fae. Agr. ttokkaido Univ., 55: 133. (115) Kim, Y. K., M. Yaguchi, and D. Rose. 1969. Isolation and amino acid composition of para-K-casein. J. Dairy Sei., 52: 316. (116) Knight, C. S. 1967. Some fundamentals of ion-exchange-cellulose design and usage in ]3iochemistry. Adv. Chromatog., 4: 61. (117) Kolar, C. W., and J. R. ]3runner. 1970. Protease-peptone fraction of bovine milk: Lacteal serum components 5 and 8-casein-

1739

(100)

(118)

(119)

(120)

(121)

(122)

(123)

(124)

(125)

(126)

(127)

(128)

(129)

(i30)

(131)

(132) (133)

associated glycoproteins. J. Dairy Sci., 53: 997. Kopfler, F. C., R. F. Peterson, and C. A. Kiddy. 1969. Amino acid composition of chromatographically separated f~-caseln A 8. J. Dairy Sci., 52: 1573. Kuwata, T., R. Niki, and S. Arima. 1969. Action of rennin on casein. Compositions and properties of glyeo-macropeptides from a-casein. J. Agr. Chem. Soe. Japan. 43: 183. Lefranc, G., and K ttan. 1967. Isolation and purification of 2 alkaline phosphatase fractions from cow's milk. Ann. Inst. Pasteur Little. 18: 185. Lira, T. /-I., W. L. Dunkley, and R. L. Merson. 1971. Role of protein in reverse osmosis of cottage cheese whey. J. Dairy Sci., 54: 306. Lingquist, ]3. 1962. Preparative separation of high molecular peptides of cheese. XVI. Int. Dairy Congr. Copenhagen Proc., p. 673. Lontle, R., and G. Preaux. 1967. Etude par Chromatographie sur Sephadex de t'Influence du Traitment thermique sur les Proteins du Serum de Lair. 36th Int. Congr. Ind. Chem., Proc., p. 820. Mackinlay, A. G., and R. G. Wake. 1965. Fractionation of S-carboxymethyl-K-casein and characterization of the components. ]31oehim. ]3iophys. Acta, 104: 167. Maki, Z., and M. Kanamori. 1969. Studies on glycopeptides from a bovine immune lactoglobulin of co]ostrum. Eiyo To Shokuryo, 22 : 42. Malpress, F. It., and M. Seid-Akhavan. 1966. Studies on human a- and K-casein fractions and human caseinoglycomacropeptide. ]3iochem. J., 101: 764. Manson, W. 1962. The effect upon casein of aqueous solutions of urea. ]3iochim. ]3iophys. Acta, 63:515. Marier, J. R., and D. Rose. 1964. Determination of cyanate, and a study of its accumulation in aqueous solutions of urea. Anal. ]3ioehem., 7: 304. Masson, P. L., and J. F. Heremans. 1968. Metal-combining properties of human lactoferrin (red milk protein). I. The involvement of bicarbonate in the reaction. European J. Biochem., 6: 579. Matoba, T., C. Nagayasu, R. Hayashi, and T. ttata. 1969. ]3itter peptides in tryptic hydrolysate of casein. Agr. ]3io]. Chem., 33 : 1662. McCabe, E. M., J. R. ]3runner, and H. A. Lillevik. 1969. Purification of para-K-casein by gel electrophoresis. J. Dairy Sci., 52: 1093. McKenzie, H. A. 1967. Milk proteins. Advances in Protein Chemistry, 22: 55. McKenzie, H. A. 1971. Milk proteins. Chemistry and Molecular Biology. Vol. IX. Academic Press. New York City. J. DAIRY SCIENCE VOL. 54, N0. 12

1740

rAOUOHI AND ROSE

(134) Mellors, A.

(135)

(136)

(137)

(138)

(139) (140) (141) (142)

(143)

(144)

(145) (146)

(147)

(148)

(149)

1969. The purification and properties of an amino acid arylamidase from bovine milk. Canadian J. Biochem., 47 : 173. Mellors, A., and V. R. Harwalkar. 1968. Glycosidases in bovine milk: a-mannosidase and its inhibition by zwitterions. Canadian J. Bioehem., 46: 1352. Mercier, J. C., J. L. Maubais, S. Poznanski, and B. Ribadeau-Dumas. 1968. Fractionnement P r e p a r a t i f des Caseines de a c h e et de Brebis par Chromatographi dur D E A E Cellulose, en Milieu Uree et 2-Mercaptoethanot. Bull. Soc. Chim. Biol., 50: 521. Mikami, :R., N. Niki, and S. Arima. 1968. Study on tile rennin-treated whole and kappa casein by diethylaminoethyl cellulose column chromatography and starch ge]electrophoresis. Japanese J. Zootech. Soc., 39 : 255. Montreui], J., J. Tonnelat, and S. Mullet. 1960. Preparation et Properties de la Lactosiderophiline (Lactotransferrine) du Lait de Femme. Biochim. Biophys. Acta, 45 : 413. Morr, C. V. 1967. Characterization of a liquid-density protein fraction fronl skimmilk. J. Dairy Sei., 50:948. Morr, C. V. 1967. Effect of urea upon the physical properties of fl-lactoglobulins A and B. J. Dairy Sci., 50: 1752. Morr, C. V. 1969. Protein aggregation in conventional and ultra high-temperature heated skimmilk. J. Dairy Sci., 52: 1174. Morr, C. V., S. T. Coulter, and R. Jenness. 1968. Comparison of column and centrifugal Sephadex method for fractionating whey and skiulmilk systenls. J. Dairy Sci., 51: 1155. Morr, C. V., and R. V. Josephson. 1968. Fractionation of skimmilk casein micelles by Sepharose column chromatography and sucrose gradient centrifugation. Abstr. J. Dairy Sci., 51: 943. Morr, C. V., and 1~. V. Josephson. 1968. Effect of calcium, N-ethylmaleimide and casein upon heat-induced whey protein aggregation. J. Dairy Sci., 51:1349. Morr, C. V., and D. B. Kenkare. 1964. On the heterogeneity of fl-lactoglobulin. J. Dairy Sci., 47: 294. Morr, C. V., D. B. Kenkare, and I. A. Gould. 1964. Fractionation of skimmilk proteins by Sephadex gel filtration. J. Dairy Sci., 47: 621. Morr, C. V., M. A. Nielsen, and S. T. Coulter. 1967. Centrifugal Sephadex procedure for fractionation of concentrated skimmilk, whey, and similar biological systems. J. Dairy Sci., 50: 305. Morr, C. V., M. A. Nielsen, and S. H . C . Lin. 1969. Sephadex equilibrium-diffusion technique for fractionating whey and skimmilk systems. J. Dairy Sci., 52: 1552. Morrison, M., H. B. Hamilton, and E.

(150) (151)

(152)

(153)

(154)

(155)

(156)

(157)

(158)

(159)

(160)

(161)

(162)

(163)

Stotz. 1957. The isolation and purification of lactoperoxidase by ion exchange chromatography. J. Biol. Chem., 228: 767. Morrison, M., and D. E. Hultquist. 1963. Lactoperoxidase. II. Isolation. J. Biol. Chem., 238: 2847. Murphy, F. A., O. Aalund, J. W. Osebold, and E. J. Carroll. 1964. Gamma-globulins of bovine lacteal secretion. Arch. Biochem. Biophys., 108: 230. Murphy, F. A., J. W. Osebold, and O. Aalund. 1965. Physical heterogeneity of bovine 5'-globulins : Characterization of 5'-M and 7-G globulins. Arch. Biochem. Biophys., 112: 126. Murphy, R. F., W. K. Downey, and I. D. Kearney. 1969. Dissociation of casein micel]es to soluble casein complexes. Abstr. Biochem. J. 115: 22. Nagasawa, T., T. Ryoki, I. Kiyosawa, and K. Kuwahara. 1967. Studies on human casein. I. Fractionationation of human casein by diethylaminoethyl cellulose column chromatography. Arch. Biochem. Biophys., 121: 502. Nagasawa, T., T. Ryoki, and I. Kiyosawa. 1966. Studies on the proteins of human milk. V I I I . Isolation and characterization of hmnan milk B~ A-globulin. Agr. Biol. Chem., 30: 693. Nagasawa, T., I. Kiyosawa, and K. Kuwahara. 1970. H a m a n casein. II. Isolation of human fl-casein fraction and human flcasein B. J. Dairy Sci., 53: 136. Nagasawa, T., I. Kiyosawa, H. Asauchi, and K. Kuwahara. 1970. Fractionation of human milk whey protein by acrylamide gel electrophoresis and gel filtration. J. Agr. Chem. Soc. Japan, 44: 89. Nagasawa, T., I. Kiyosawa, H. Asauchi, and K. Kuwahara. 1970. Acrylamide gel electrophoresis of human casein and amino acid compositions of human casein fractions obtained by DEAE-cellulose column chromatography. J. Agr. Chem. Soc. Japan, 44: 136. Nakai, S., H. K. Wilson, and E. O. Herreid. 1966. Preferential oxidation of tryptophan residues in K-casein with ~-bromosuccinimide. J. Dairy Sei., 49:469. Nakai, S., H. K. Wilson, and E. O. Herreid. 1966. Effect of alkalization, oxidation, reduction and storage on elation patterns of K-casein obtained by gel filtration. J. Dairy Sci., 49: 1331. Nakai, S., H. K. Wilson, and E. O. Herreid. 1966. Calculation of the molecular size of K-casein fractions obtained by gelfiltration a f t e r storage and rennin action. J. Dairy Sci., 49: 1557. Nakai, S., W. A. Blair, A. Hehnersen, and B. A. Eagles. 1968. Desa]ting milk and whey by ion retardation and gel filtration. J. Dairy Sci., 51:1909. Nakanishi, T., and T. Itoh. ]969. Studies

J'. D A I I ~ r SCIENCE VOL. 5 4 , NO. 1 2

PROTEII~

SEPARATION

1741

(164)

(165)

(166)

(167)

(168) (169)

(170)

(171)

(172)

(173)

(174)

(175)

(176)

(177) (178)

on changes of the milk casein by various treatments. VI. Changes of Sephadex gel filtration p a t t e r n and sialic acid content in casein solution by heat treatment. J. Agr. Chem. Soc. Japan, 43:306. Nakanishi, T., and T. Itoh. 1969. Studies on the changes of the milk casein by various treatments. V I I I . On the properties of casein flocculated during frozen storage of milk and mechanism for flocculation. J. Agr. Chem. Soe. Japan, 43: 725. Nakanishl, T., and T: Itoh. 1970. Studies on the changes of the milk casein by various treatments. X. Changes of the K-casein solution by heat treatment. J. Agr. Chem. Soc. Japan, 44: 118. Nakanishl, T., K. Takahashi, and T. Imagawa. 1968. Studies on changes of whey protein by heat treatment. II. Interaction of fl-lactoglobulin, a-lactalbumin and Kcasein. Japanese J. Dairy Sei., 17:A28. Nakanishi, T., K. Takahashi, and T. Itoh. 1967. Changes in whey protein on heating. I. Changes in whey protein solution and skimmilk. Japanese J. Dairy Sci., 16: A l l 3 . Nicholas, R. A., and J. B. Fox, Jr. 1969. Continuous chromatography apparatus. I I I . Application. J. Chromatogr., 43: 61. Nikl, R., and S. Arima. 1969. Influence of p i t on the temperature-dependent polymerization of fl-casein. Agr. Biol. Chem., 33: 826. Nishikawa, I., N. Abe, and K. Saito. 1966. Changes of bovine milk protein by heating as revealed by DEAE-eellu]ose column chromatography. J. Agr. Chem. Soc. J a p a n , 40 : 414. Nishikawa, I., N. Murata, It. Yoshida, and K. Salto. 1969. Studies on human milk proteins. II. Properties of human casein (2). J. Agr. Chem. Soc. Japan, 4 3 : 5 0 . N]shikawa, I., and K. Saito. 1969. Studies on human milk proteins. I. Properties of human casein (1). J. Agr. Chem. Soc. Japan, 43 : 45. Noble, R. W., Jr., and D. F. Waugh. 1965. Casein micelles. Formation and structure. I. J. Amer. Chem. Soe., 87: 2236. Oram, Y. D., and B. Reiter. 1968. Inhibition of bacteria by lactoferrin and other iron-chelating agents. Biochim. Biophys. Acta, 170: 351. Parry, R. M., Jr., R. C. Chandan, and K. M. Shahani. 1969. Isolation and charaeterization of human milk lysozyme. Arch. Biochim. Biophys., 130: 59. Parry, R. M., Jr., L. W. Ford, and R. J. Carroll. 1969. Interaction of a~i and ~casein in the absence of calcium ions. Abstr. J. Dairy Sci., 52: 902. Payens, T. A. J. 1961. Zone electrophoresis of casein in urea-buffer mixture. Bioclaim. Biophys. Acta, 46: 411. Peterson, E. A., and H. A. Sober. 1956. Chromatography of proteins. I. Cellulose

(179) (180)

(181) (182) (183) (184)

(185)

(186)

(187) (188) (189)

(190)

(191)

(192)

(193)

(194)

(195)

ion-exchange adsorbents. J. Amer. Chem. Soc., 78: 751. Peterson, E. A., and H. A. Sober. 1959. Variable gradient device for chromatography. Anal. Chem., 31: 857. Peterson, E. A., and H. A. Sober. 1960. Chromatography of the plasma proteins. "The Plasma Proteins". ol. 1. p. 105, ed., F. W. Putnam. Academic Press, New York City. Peterson, E. A., and It. A. Sober. 1961. Column chromatography of proteins: Substituted celluloses. Methods Enzymol., 5 : 1 . Peterson, R. F. 1969. Electrofocussing of milk proteins. Abstr. J. Dairy Sci., 52: 903. Pharmacia Fine Chemicals AB. ]968. Industrial gel filtration with the Sephamatic system. Uppsala, Sweden. Phinips, A. W., and P. G. Gibbs. 1961, Techniques for the fractionation of microbiologically active peptides derived from casein. Bioehem. J., 81: 551. Piez, K. A., E. W. Davie, J. E. Folk, and J. A Glander. ]961. fl-Lactoglobulins A and B. I. Chromatographic separation and amino acid composition. J. Biol. Chem., 236 : 2912. Polis, B. D., and H. W. Shmukler. 1953. Crystalline ]actoperoxidase. I. Isolation by displacement chromatography. II. Physiochemical and enzymatic properties. J. Biol. Chem., 201: 475. Porath, J. 1960. Gel filtration of proteins, peptides and amino acids. Biochim. Biophys. Acta, 39: 193. Porath, J. 1962. Cross-linked dextrans and molecular sieves. Adv. Protein Chem., 17: 299. Porath, J., and P. Flodin. 1959. Gel filtration: a method for desalting and group separation. Nature, 183: 1657. Preaux, G., and R. Lontie. 1961. Fractionation of proteins from milk serum on a column of Sephadex. Arch. Int. Physiol. Biochem., 69: 100. Preaux, G., and R. Lontie. 1961. Fractionation of milk whey proteins on Sephadex G-75 co]unms. Protides Biol. Fluids. 9: 105. Preaux, G., and R. Lontie. 1966. Chromatography on Sephadex of fl-lactoglobulin A and B with native and blocked thio] groups. Protides Fluids, 14: 611. Preaux, G., P. Weer, E. Peirsman, K. Tielemaus, and R. Lontie. 1965. Separation of three compounds immunologically identical with a-lactalbumin from milk serum. Archiv. Physiol. Biochcm., 37: 154. Pujolles, J., B. Ribadeau-Dumas, J. Garnier, and R. Pion. 1966. A study of Kcasein components. I. Preparation. Evidence for a common C-terminal sequence. Biochem. Biophys. Res. Comm., 25: 285. Purkayastha, R., M. Yaguchi, J. R. Marier, and D. Rose. 1967. Distribution of sialic
J . DAIRY SCIENCE YOL. 54, NO. 12

1742

YAGUCHI AND ROSE acid among r-casein components. Abstr. J. Dairy Sei., 50: 940. Ribadeau-Dumas, B. 1961. Fractionncment de la caseine par chromatographle sur colonne de diethylaminoethyl-cellulose en milieu uree. Eiochim. Eiophys. Acta, 54: 400. 1%ibadeau-Dumas, B., J. L. Maubois, G. Maequot, and J. Garnier. 1964. Etude de la constitution de ]a caseine de vache par chromatographic sur colonnes de diethylaminoethyl-cellulose en milieu uree. Biochim. Biophys. Acta, 82: 494. Rombauts, W. A., W. A. Schroeder, and M. Morrison. 1967. I~ovine laetoperoxidase. Partial characterization of the further purified protein. Biochemistry, 6:2965. Rose, D., J. R. Brunner, E. Kalan, B . L . Larson, P. Melnychyn, tI. E. Swaisgood, and D. F. Waugh. 1970. Nomenclature of the proteins of cow's milk. Third revision. J. Dairy Sci., 53:1. Rose, D., D. T. Davies, and M. Yaguchi. 1968. Quantitative deternlination of the major components of casein mixtures by column chromatography on DEAE-cellulose. J. Dairy Sci., 51: 8. Rose, D., and J. R. ~[arier. 1963. Fractionation of r-casein on a diethylmuinoe~dlyl ec]h l o s e column. J. Dairy Sci., 46: 1323. Saito, Z., and Y. tIashimoto. 1963. The milk ]ipases. V. Effect of surface-active agents and ethylene-diaminetetraaeetate on lipases and purification of lipases by chromatography on anion-exchange cellulose. Japanese J. Zootechnlcal Sci., 34: 393. Samuelsson, E. G., P. Tibbling, and S. Holm. 1967. Gel filtration--road to new products. Protein-enriched milk: one upp]ication. Food Teehnol., 21:1535. Sawyer, W. H. 1968. Heat denaturation of bovine fl-lactoglobulins and relevance of disulfide aggregation. J. Dairy Sci., 51: 323. Schmidt, D. G., P. Both, and P. J. De Koning. 1966. Fractionation and some properties of r-casein variants. J. Dairy Sci., 49 : 776. Schober, R., and i'. Heimburger. 1960. Trennung yon Na-caseinat durch Saulenchromatographie an Ionenaustauscher-Cellulose. Milchwissenehaft, 15: 607. Schober, R., N. Heimburger, and D. Enkelmann. 1959. Trennung yon Melkenproteinen durch Saulenchronmtographle an Ionenaustrauscher-cellulose. Milchwissenschaft, 14: 432. Sell, S. 1967. Isolation and characterization of rabbit colostral IgA. Immunochemistry, 4: 49. Shahani, K. M. 1966. Milk enzymes; Their role and significance. J. Dairy Sci., 49: 907. Shanina-Vagina, V. I., and L. S. Edvabnaya. 1967. Use of gel filtration for characterizing casein and its hydrolyzates. Laboratornee delo. 67: 670. Dairy Sei. Abstr., 31: 224 (1969). (211) Sober, tI. A., 1%. W. ttartley, Jr., W. R. Carroll, and E. A. Peterson. 1965. Fractionation of proteins. The Proteins, 3: 1. (212) Sober, H. A., and E. A. Peterson. 1954. Chromatography of protcins on cellulose ion exchangers. J. Amer. Chem. See., 76:
1711.

(196)

(197)

(198)

(199)

(200)

(201) (202)

(203)

(204)

(205)

(206)

(207)

(208)

(209) (210)

(213) Sober, tt. A., and E. A. Peterson. 1968. Protein chromatography on ion exchange cellulose. Federation Prec., 17: 1116. (214) Sober, H. A., and E. A. Peterson. 1960. Chromatographic evaluation of protein mixture. In Amino Acids, Proteins and Cancer Biochemistry, ed., Edsall. Academic Press, New York City. P. 61. (215) Szuchet-Derechin, S., and P. Johnson. 1965. The "albumin" fraction of bovine milk. :[. Overall chromatographic fractionation on DEAE-cellul0se. European Polymer J., 1: 271. (216) Szuchet-Derechin, S., and P. Johnson. 1965. The "albumin" fraction of bovine milk. II. The isolation and physico-chemica] properties of the red proteins. European Polymer" J., 1: 283. (217) Szuchet-Derechin, S., and P. Johnson. 1966. The "albumin" fraction of bovine milk. I I I . The micro-heterogeneity of the red protein. European Polymer J., 2: 29. (218) Sznehet-Derechin, S., and P. Johnson. ]966. The "albumin" fraction of bovine milk. IV. A physico-chemical study of r e d p r o t e i n A. European Polymer J., 2: 115. (219) Tarassuk, N. P., and M. Yaguchi. 1962. Chromatography of milk proteins on D E A E cellulose. J. Dairy Sci., 45: 253. (220) Tarassuk, N. F., M. Yaguchi, and J. B. Cal]is. 1965. Effect of temperature on the composition of casein fractions eluted from DEAE cellulose colmnn. J. Dairy Sci., 48: 606. (221) Tessler, H., M. Yaguehi, and D. 1%ose. 1969. Zonal ultracentrifugation of fl-lactoglobulin and r-casein complex induced by heat. J. Dairy Sci., 52: 139. (222) Thompson, M. P. 1966. DEAE-cclluloseurea chromatography of casein in the presence of 2-mercaptoethanol. J. Dairy Sci., 49 : 792. (223) Thompson, M. P., W. G. Gordon, 1%. T. Boswell, and I-I. M. Farrell, Jr. 1969. Solubility solvation and stabilization of asland fl-caseins. J. Dairy Sci., 52: 1166. (224) Thompson, M. P., and C. A. Kiddy. 1964. Genetic polymorphism in caseins of cow's milk. I I I . Isolation and properties of a~caseins A, B, and C. J. Dairy Sci., 47: 626. (225) Thompson, M. P., and L. Fcpper. 1964. Genetic polymorphism in caseins of cow's milk. IV. Isolation and properties of flcasein A, B, and C. J. Dairy Sei., 47: 633. 1968. (226) Tokita, F., and F. Takahashi. Changes in heat-treated nlilk proteins and

J. DAIRY SCIENCE VOL. 54, NO. 12

e~0TEIS SEPARATION

1743

(227) (228) (229) (230)

(231)

(232)

(233)

(234)

revealed by immunoelectrophoresis and other methods. Japanese J. Zootech. Sci., 39: 266. Tran, . D., and B. E. Baker. 1970. Casein. IX. Carbohydrate moiety of K-casein. J. Dairy Sei., 53: 1009. Tripathi, K. K., and C. W. Gehrke. 1969. Chromatography and characterization of gamma-casein. J. Chromatogr., 43:322. Tripathi, K. K., and C. W. Gehrke. 1970. Chemical and chromatographic isolation of kappa-casein. J. Chromatogr., 46: 280. Uusi-Rauva, E., K. Kiura, and M. Antila. 1969. Die Fraktionierung des Kaseines der Milch durch die DEAE-Zcllulose-ionenaustrausch-Chromatographie. Suomen Kemistilehti, 42B: 371. Uusi-Rauva, E., R. Pajula, and M. Antila. 1969. Gel filtration of the whey proteins of milk. Suomen Kemistilehti, 42B: 328. Wa]]ander, J. F., and A. M. Swanson. 1968. Distribution of the ml]k lipase system in unheated and heated skimmilk. Abstr. J. Dairy Sci., 51: 941. Waugh, D. F., M. L. Ludwig, J. M. Gillespie, B. Melton, M. Foley, and E. S. Kleiner. 1962. The a~-caseins of bovine milk. J. Amer. Chem. Soc., 84: 4929. Waugh, D. F., L. K. Creamer, C. W. Slattery, and G. W. Dresdner. 1970. Core polymers of casein micelles. Biochemistry, 4:

7s6.
(235) Whitaker, J. R. 1963. Determination of molecular weights of proteins by gel filtration on Sephadex. Anal. Chem., 35: 1950. (236) Windle, J. J., A. K. Wisersema, J. R. Clark, and R. E. Feeney. 1963. Investigation of the iron and copper complexes of avian conalbumins and human transferrins by electron paramagnetic resonance. Biochemistry, 2 : 1341.

(237) Woychik, J. H., E. B. Kalan, and M. E. Noelken. 1966. Chromatographic isolation and partial characterization of reduced Kcasein components. Biochemistry, 5: 2276. (238) Woychik, J. H., and M. V. Wondolowski. 1967. Isolation of bovine para-K casein components. Amer. Chem. Soc., 154th Meet. Abstr. C-14. (239) Woychik, J. H., and M. V. Wondolowski. 1969. Chromatographic isolation and amino acid composition of sow a-, fl-, and Kcaseins. J. Dairy Sci., 52: 901. (240) Yaguchi, M., D. T. Davies, and Y. K. Kim. 1968. Preparation of K-casein by gel filtration. J. Dairy Sei., 51: 473. (241) aguchi, M., and N. P. Tarassuk. 1967. Gel filtration of acid casein and skimmilk on Sephadex. J. Dairy Sci., 50: 1985. (242) aguchi, M., N. P. Tarassuk, and N. Abe. 1964. Distribution of tipase in milk proteins. I. D E A E cellulose column chromatography. J. Dairy Sci., 47: 1167. (243) aguehi, M., N. P. Tarassuk, and H. G. Hunziker. 1961. Chromatography of milk proteins on anion-exchange cellulose. J. Dairy Sci., 44: 589. (244) Yamauchi, t_., Y. ttonma, and T. Tsugo. 1967. Separation of soluble calcium phosphate and citrate in milk by Sephadex gel filtration. Japanene J. Zootech. Sci., 38: 117. (245) Woshida, S., S. Arima, and Y. Hashimoto. 1965. A study of casein micelle. Fractionation of proteins in casein micelle. J. Agr. Chem. Soc. Japan, 39: 71. (246) Zittle, C. A. 1960. Column chromatography of casein on the adsorbent diethylaminoethyl (DEAE) cellulose. Abstr. J. Dairy Sci., 43 : 855. (247) Zittle, C. A. 1965. Purification of protease in cow's milk. g. Dairy Sci., 48: 771.

J. DAIRY SUIENeE VOL. 54, NO. ]2