SYMPOSIUM: GENETIC PERSPECTIVES ON MILK PROTEINS: COMPARATIVE STUD1 ES AND NOMENCLATURE Review and Update of Casein ChemIstryl,* HAROLD E. SWAISGOOD Southeast Dairy Food Research Center Department of Food Science North Carolina State University Raleigh 276957624 ABSTRACT Of all food proteins, bovine milk pro- teins are probably the most well charac- terized chemically, physically, and ge- netically. The primary structures are known for most genetic variants of asl-, as2-, 0-. and K-caseins, &lactoglobulin, and a-lactalbumin. Secondary and ter- tiary structures of the whey proteins have been determined, and secondary struc- tures of the caseins have been predicted from spectral studies. The caseins, al- though less ordered in structure and more flexible than the typical globular whey proteins, have significant amounts of secondary and, probably, tertiary structure. The amphipathic structure of the caseins is especially noteworthy; thus, these proteins most likely are divided into polar and hydrophobic do- mains. The presence of anionic phos- phoseryl residue clusters in the calcium- sensitive casein polar domains is particu- larly significant because of their interac- tion with calcium ions, or calcium salts, or both, and the formation of micelles. Flexibility of casein structures is reflected by their susceptibilities to limited proteolysis, which dramatically changes functionality. Received August 24, 1992. Accepted December 16, 1992. 'Paper Number FSR92-27 of the Journal Series of the Department of Food Science, Noah Carolina State Univer- sity, Raleigh 27695-7624. The use of trade names in this publication does not imply endorsement by the North Carolina Research Service of products named or criticism of similar ones not mentioned. 2Support for the presentation of this symposium papcr was paaially provided by che California Dairy Foods Research Center. (Key words: casein chemistry, milk pro- teins, nomenclature, symposium) Abbreviation key: FPLC = fast protein liquid chromatography. INTRODUCTION A great opportunity for increased utilization of milk rests with increased use of milk pro- teins as highly functional food ingredients. Functional properties are directly related to the physicochemical characteristics of the protein. Optimal functionality requires different phys- icochemical properties for specific functions, such as water binding, gelation, emulsification, and foaming. Therefore, an understanding of the relationship between structure and func- tionality will be essential for selection of in- gredients and ultimately for design of function- ality by bioprocessing operations or genetic modification. Many of the physicochemical characteristics of individual milk proteins are now known, and much of the current research is focused on understanding the protein inter- actions that occur among themselves, with other proteins, and with other food ingredients. The brief review presented herein summa- rizes some areas of milk protein chemistry that provide the basis for future investigation of the relationship between structural and phys- icochemical characteristics and functionality. A number of reviews have appeared previously (6, 50, 51). Therefore, the present article at- tempts to summarize several fundamental characteristics and to review a few more recent studies. IDENTIFICATION. COMPOSITION, QUANTITATION, .AND ISOLATION OF CASEINS This review uses the revised nomenclature recommended by the ADSA milk protein 1993 J Dairy Sci 76:3054-3061 3054
Transcript
SYMPOSIUM: GENETIC PERSPECTIVES ON MILK PROTEINS: COMPARATIVE STUD1 ES AND NOMENCLATURE
Review and Update of Casein ChemIstryl,*
HAROLD E. SWAISGOOD Southeast Dairy Food Research Center
Department of Food Science North Carolina State University
Raleigh 276957624
ABSTRACT
Of all food proteins, bovine milk pro- teins are probably the most well charac- terized chemically, physically, and ge- netically. The primary structures are known for most genetic variants of a s l - , as2-, 0-. and K-caseins, &lactoglobulin, and a-lactalbumin. Secondary and ter- tiary structures of the whey proteins have been determined, and secondary struc- tures of the caseins have been predicted from spectral studies. The caseins, al- though less ordered in structure and more flexible than the typical globular whey proteins, have significant amounts of secondary and, probably, tertiary structure. The amphipathic structure of the caseins is especially noteworthy; thus, these proteins most likely are divided into polar and hydrophobic do- mains. The presence of anionic phos- phoseryl residue clusters in the calcium- sensitive casein polar domains is particu- larly significant because of their interac- tion with calcium ions, or calcium salts, or both, and the formation of micelles. Flexibility of casein structures is reflected by their susceptibilities to limited proteolysis, which dramatically changes functionality.
Received August 24, 1992. Accepted December 16, 1992. 'Paper Number FSR92-27 of the Journal Series of the
Department of Food Science, Noah Carolina State Univer- sity, Raleigh 27695-7624. The use of trade names in this publication does not imply endorsement by the North Carolina Research Service of products named or criticism of similar ones not mentioned.
2Support for the presentation of this symposium papcr was paaially provided by che California Dairy Foods Research Center.
Abbreviation key: FPLC = fast protein liquid chromatography.
INTRODUCTION
A great opportunity for increased utilization of milk rests with increased use of milk pro- teins as highly functional food ingredients. Functional properties are directly related to the physicochemical characteristics of the protein. Optimal functionality requires different phys- icochemical properties for specific functions, such as water binding, gelation, emulsification, and foaming. Therefore, an understanding of the relationship between structure and func- tionality will be essential for selection of in- gredients and ultimately for design of function- ality by bioprocessing operations or genetic modification. Many of the physicochemical characteristics of individual milk proteins are now known, and much of the current research is focused on understanding the protein inter- actions that occur among themselves, with other proteins, and with other food ingredients.
The brief review presented herein summa- rizes some areas of milk protein chemistry that provide the basis for future investigation of the relationship between structural and phys- icochemical characteristics and functionality. A number of reviews have appeared previously (6, 50, 51). Therefore, the present article at- tempts to summarize several fundamental characteristics and to review a few more recent studies.
This review uses the revised nomenclature recommended by the ADSA milk protein
1993 J Dairy Sci 76:3054-3061 3054
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%I
nomenclature report (20). The tenacity of pro- tein interactions among the caseins requires use of a dissociative agent to identify or to isolate individual proteins. Historically, caseins have been identified by alkaline urea-PAGE (20, 50). More recently, ion-exchange chro- matography in urea has proven useful for iden- tification, isolation, or quantitation of in- dividual caseins. Following initial studies of fractionation by urea-DEAE-cellulose chro- matography (15, 47, 48, 5 3 , procedures for fractionation and quantitation have been devel- oped using HPLC (29) and fast protein liquid chromatography (FPLC) (3, 13, 25) types of chromatographic supports. For example, the fractionation of caseins by anion-exchange chromatography, as achieved by Barrefors et al. (3) using Mono Q FPLC, is illustrated in Figure 1; fractionation by cation-exchange chromatography, developed by Hollar et al. (25) using Mono S FPLC, is shown in Figure 2. Resolution of individual proteins is quite good, although separation of genetic variants in a complex sample is probably not possible. Quantitation of individual caseins is very good by either anion-exchange or cation-exchange
xs2
.4
.3
p 2
I
IO 20 ELUTION VOLUME (mi)
Figure 1. Fast protein liquid chromatography ion- exchange chromatography of acid-precipitated castin (CN) obtained from an individual cow (genotype: &N A; p- CN A2; ctSl-CN 8). Chromatography was performed with a Mono Q column (5 x 50 mm) using a flow rate of 60 mVh. Taken from Bamfors et al. (3) with permission.
1.v ' ' 1 A I B ? l r 4
0 - rilEe,n
s G 5 z
9 18 27 36 45 0 0
ELUTION VOLUME (ml)
Figure 2. Elution profile of herd bulk whole casein obtained by fast protein liquid chromatography at 20'C using a Mono S HR5/5 column. Protein was eluted with urta-acetate buffer @H 5% .02 M acetate; 6 M m) and an NaCl gradient. About 2 to 5 mg of protein were applied to the column, and the flow rate was 1 ml/min. Absor- bance (-); NaCl gradient (-). Taken from Hollar et al. (2s) with permission.
chromatography, as demonstrated by the excel- lent correlation between these methods for de- termination of q-, aa-, &, and K-caseins (25). The correlations are best for asl- and 6- caseins because these are the best resolved. Determination of K-casein is also complicated by incomplete resolution of some of the glycosylated forms from other proteins. Nevertheless, using crude preparations of whole K-casein as the starting material, 9 to 10 of the posttranslationally modified components can be resolved by DEAE-cellulose chro- matography (54, 59).
Ion-exchange materials also have been used for relatively large-scale fractionation of caseins. For example, 13 g of whole casein were fractionated into relatively pure asl-, P-, and K-caseins using a QAE Zeta Prep 250 cartridge (40). However, crS2-casein could not be resolved from a,l-casein. Such studies sug- gest that, in the future, large-scale fractionation may be possible using improved ion-exchange materials.
CHARACTERISTICS OF CASEIN STRUCTURES
Prlmary Structures
The primary structures of most of the known genetic variants of each casein have now been well established by chemical se-
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quence and by cDNA or genomic DNA se- quences. The latest corrections have been presented in a recent review (51). These revi- sions allow more accurate compositions and characteristics to be calculated. Composition for the major variants of the four individual caseins is listed in Table 1. The unique fea- tures of these compositions compared with those of typical globular proteins are the pres- ence and numbers of phosphoseryl residues and the high frequency of prolyl residues. Be- cause of absence of cysteinyl residues in asl- and &casein, these proteins cannot participate in sulfhydryl-disulfide interchange crosslinking reactions. Accurate compositions also permit calculation of a number of physicochemical parameters, such as the molecular charge characteristics given in Table 2. Because the tertiary structures of caseins are apparently more flexible than those of typical globular proteins, calculated values agree well with those measured experimentally (Table 2). For example, their rates of electrophoretic migra- tion and order of ion-exchange chromato-
graphic elutions, with a few exceptions, are consistent with their net charges.
A unique feature of the primary structures of caseins, which are most likely responsible for their unique functional properties, is the distinct amphipathic nature of their sequences. These structures suggest that their tertiary structures are organized into polar and hydrophobic domains (50). Furthermore, the polar domains of the calcium-sensitive caseins contain anionic clusters comprising phos- phoseryl residues, but the polar domain of K- casein, although strongly anionic, does not have phosphoseryl residue clusters. In addition to determination of the solubility in the pres- ence of calcium ion, t h i s structural characteris- tic is undoubtedly responsible for the unique interactions with calcium salts that define micelle structure.
Secondary Structure8
Because a direct observation of secondary structures of caseins by X-ray crystallography
TABLE 1. Chemical composition of the commonly Occurring caseins (CN).'
a,l-CN Q s 2 - a K-CN &CN Acid B-8P A-1lP B-1P AZ-5P
'Calculated using the relation ZH = hh - r, where f = ci (IljkjhHY(1 + k,hH). and ZH is the net p t O ~ C charge. The apparent pKj used were a-carboxyl, 3.6 (41); phosphoseryl, pK1 = 1.5, pK2 = 6.4 (10); 8-, y-carboxyl, 4.9 (23); histidyl. 6.6 (41); and a-amino, 7.4 (41) except for x-casein, where the more common value for b-, y-carboxyl of 4.6 was used (41). If an apparent pK of 4.9 is used, the isoionic point is 5.84 for K-CN A and 6.06 for K-CN B.
Talcdated as for charge at pH 6.6, but Z, = 0 at the isoionic pH.
is not possible, the amount of the various structures in caseins has been estimated from measurements using various spectral tech- niques and using algorithms to predict secon- dary structure from the established primary
TABLE 3. Secondary structure of the caseins (o.
structures (Table 3). These results suggest that the Erequent statement, that caseins lack secon- dary structure, is incorrect (50). Furthermore, prediction methods suggest that several struc- tural motifs may be present [e.g., &YO structure in the hydrophobic domain of K-casein (45) and a turn-8-strand-turn motif in the chymosin- sensitive region (28)]. Also, the calcium- sensitive caseins may contain helix-loop-helix motifs centered on the sites of phosphorylation (28).
Tertiary Structures
Obviously, the three-dimensional structures of the caseins are not known because caseins apparently are not crystallizable. Nevertheless, recent attempts have been made to predict the tertiary structures of caseins from their primary structures by molecular modeling (33, 34). The predicted structures are compatible with the gross features of division into hydrophobic and polar domains as predicted from their am- phipathic primary structures and their phys- icochemical properties (50). The predicted structures suggest that the hydrophobic do- mains of individual caseins may interact through formation of extended secondary structures leading to formation of submicelles. Physicochemical properties indicate that the tertiary structures of these proteins are more open and flexible than those of typical globular proteins (50). Greater flexibility than that of typical globular proteins is also indicated by analysis of proteolysis rates (9, 52). The high frequency of prolyl residues may provide an
'Circular dichroism. 2Numbers in parentheses are the reference numbers. 30btained by analysis of optical rotatory dispersion data.
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architectural stiffness, yielding an overall open structure with typical flexibility around in- dividual residues and a rapidly fluctuating secondary structure. Thus, caseins may exhibit a higher structural motility than typical globu- lar proteins.
PRODUCTS OF LIMITED PROTEOLYSIS OF CASEINS
Because the structures of caseins are not random coils of completely flexible chains, a certain number of susceptible residues are more rapidly hydrolyzed. As expected, these residues occur in the most flexible regions of the protein structure, which usually represents residues in turns or regions between hydropho- bic and polar domains (52). The structure of j3- casein apparently is particularly open and flex- ible in the region between the N-terminal polar domain and the C-terminal hydrophobic do- main. Thus, limited proteolysis by the natu- rally occurring proteinase plasmin (18, 19) results in the presence of y-caseins and proteose-peptones in normal milks (Table 4). Being derived from the hydrophobic domain, y-caseins are extremely nonpolar and can be extracted in organic solvents (46). However, the proteose-peptones resulting from the polar domain are highly charged and very heat sta- ble.
The ancient art of cheese making is based on the liberation of the polar domain of K-
casein by cleavage of an apparently especially flexible and exposed peptide bond joining the hydrophobic and polar domains of this mole- cule. The resulting loss of charge and increase in surface hydrophobicity lead to coagulation of micelles. Upon further proteolysis by chyrnosin during curd maturation, a flexible region between the hydrophobic N-terminal domain of a,l-casein and the polar domain is hydrolyzed, yielding cusl-casein(f25- 199) (1 2, 32). The increased hydrophilicity of this large C-terminal peptide is correlated with changes in the rheological characteristics of the curd (12).
More extensive proteolysis of the calcium- sensitive caseins by neutral or alkaline pro- teinases frequently produces bitter peptides (37, 39, 49). Such peptides usually are derived from the C-terminal, 14-residue sequence of 0- caseins (Table 4). Bioactive peptides derived by in vivo digestion of the calcium-sensitive caseins also have been obtained in recent studies (36, 38, 60, 61).
CASEIN INTERACTIONS WITH CALCIUM
Casein interactions with calcium ions and calcium salts (colloidal calcium phosphate) are necessary for formation and maintenance of casein micelles. The anionic clusters of phos- phoseryl residues are the primary sites of cal- cium binding (24, 26). Consequently, solubility
TABLE 4. Some peptides or domains derived from casein (CN) by limited protealysis.
Roteose-peptones 8-CN X(fl-105 or 107) 8-CN X(f29-105 or 107) 8-CN X(fl-28)
Plasmin 7-CN
Heat stable, very soluble Heat stable, very soluble Heat stable, very soluble
Chymosin
Chymotrypsin
Para-K-CN; K C N X(fl-105) Macropeptide; K-CN X(fl06-169) asl-CN I; ~ ~ 1 - 0 4 B C(f25-199) B - 0 4 X(fl-52) Possible amphipathic helix
Hydrophobic, low solubility Very polar, very soluble Increased hydrophilicity
Trypsin (more extensive hydrolysis) Peptides from the region of B-CN Bitter peptides X(fl96-209)
Pepsin, intestinal enzymes, or both Peptides from the region of &CN Opioid activity X(f60-70)
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of the isolated proteins as a function of Ca2+ concentration is correlated with the number of these clusters per molecule; thus, the order of solubilities is c~~2-c c~~1-c 8-c K-casein with 3, 2, 1, and 0 clusters, respectively (2, 50, 56). A common cluster sequence is SerP-SerP-SerP- GluGlu. Glutamyl residues are a part of each of the cluster sequences and are likely to be an integral part of the anionic site-binding cluster. Studies of individual caseins (14, 15, 16, 21, 42, 43, 44) and of casein submicelles (30) showed that more Ca2+ was bound than the expected number of phosphoseryl residues would indicate. Also, direct evidence of car- boxylate participation has come from infrared spectra (7, 42).
Analysis of Ca2+ binding to a,l-casein has suggested several phases in the binding equilibria as Ca2+ concentrations increased. Thus, below 1 mM Ca2+, exothermic binding (27)-primarily to phosphoseryl residues (42) accompanied by transfer of aromatic residues from an aqueous to an apolar environment (42)-is the major equilibrium. Between 1 and 3 mM Ca2+, a second exothermic phase occurs, followed by an increasingly endothermic reac- tion (27) with initiation of binding to carboxy- late residues (42) and increasing self- association (17, 27). Finally, above 3 mM Ca2+, the reaction is very endothermic (27); binding is primarily to carboxylate residues (42), accompanied by increasing aggregation (27), and eventually leads to precipitation be-
tween 5 to 6 mM Ca2+ at the usual protein concentrations. At a molecular level, these results are interpreted to suggest that initial binding causes conformational changes with increased exothermic H-bonding, perhaps with formation of extended @-sheet structures, even- tually leading to endothermic intermolecular hydrophobic interactions. Continued binding to carboxylate residues further reduces inter- molecular electrostatic repulsion, and hydrophobic interaction of the hydrophobic domains leads to formation of large ag- gregates.
The effects of pH, temperature, and ionic strength on casein solubilities and on the sta- bility of micelles are consistent with the effects on the characteristics of the binding equilibria observed with individual caseins (4, 14, 44) and, more recently, with casein submicelles (see Table 5) (30). Increased temperature in- creases the affinity, and increased pH increases the affinity and the number of sites; increased ionic strength decreases the affinity. Similarly, the solubilities of caseins in the presence of Ca2+ decrease with increasing temperature and pH and increase with ionic strength (14, 21, 44). The affinities for Ca2+ (Table 5 ) for sub- micelles are slightly higher than those previ- ously reported for individual caseins, perhaps because the submicelle-bindin equilibria were characterized below 1 mM C$+ at which only the highest affinity binding occurs (30). Alter- natively, the structure of anionic clusters in
TABLE 5. Effect of pH and temperature on the equilibrium dissociation constant and the total 45Ca bound in the analytical affinity column of immobilizcd casein submicelles.
Ca2+ ' cap042 SMUF3
cc) w (rtml) w ( W O I ) w OlmOl) 6.0 20 680 .46 670 .36 60 .43 6.7 20 88 .52 96 .61 30 .56 6.7 30 70 .44 78 .48 16 .59 6.7 40 52 .43 60 .46 9 .57 7.5 20 .8 .65 65 .88 .9 .73
*CaClz dissolved in 25 mM imidazole buffer. K m = The equilibrium dissociation constant for 45Ca2+. M: = The total amount of 45Ca-binding sites in the column of immobilized casein submicelles (42.9 m o l of casein*using a molecular mass average of 23.3 kDa.)
X a C 4 and KH2PO4 dissolved in 25 mM imidazole buffer. Ratio of PO4:Ca was maintained at 1.29. 3Simulated milk ultrafiltrate (31).
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submicelles may provide more ligand interac- tions with Ca2+.
In the presence of inorganic phosphates and other ions in milk salts, such as citrates, the interactions of calcium with casein are more complex. Such interactions are of key impor- tance to the structure and stability of natural milk micelles. Results from studies of calcium interaction with submicelles (30) in the pres- ence of dilute milk salts suggest that a higher affinity of binding occurs (Table 5). van Dijk (57, 58) has proposed the formation of ion clusters, including Ca2+, inorganic phosphate, and possibly other ions that interact with the phosphoseryl residues. Perhaps extended clusters are formed between submicelle anionic clusters and milk salt clusters that optimize liganding.
REFERENCES
1 Andrews. A. L., D. Atkinson, M.T.A., Evans, E. G. Finer, J. P. Green, M. C. Phillips. and R. N. Robert- son. 1979. The conformation and aggregation of bo- vine &casein A. Biopolymers 18:1105.
2Aoki, T., K. Toyooka, and Y. Kako. 1985. Role of phosphate groups in the calcium sensitivity of as2- casein. J. Dairy Sci. 68:1624.
3 Bamfors, P., B. Ekstrand, L. Faptrstam, M. Larsson- Raznikiewicz, J. Schaar, and P. Steffner. 1985. Fast protein liquid chromatography 0 of bovine caseins. Milchwissenschaft 40:257.
4Baumy, J. J., and G. Brule. 1988. Effect of pH and ionic strcngth on the binding of bivalent cations to @- casein. Lait 68:409.
5 Bigelow, C. C. 1967. On the average hydrophobicity of proteins and the relation between it and protein structure. J. Theor. Biol. 16:187.
6Brunner. J. R. 1981. Cow milk proteins: twenty-five ‘ears of progress. J. Dairy Sci. 64:1038.
7 kyler, D. M., and H. M. Farrell, Jr. 1989. Infrared spectroscopic evidence for calcium ion interaction with carboxylate groups of casein. J. Dairy Sci. 72: 1719.
8Byler, D. M., H. M. Farrell, Jr.. and H. Susi. 1988. Raman spectroscopic study of casein structure. J. Dairy Sci. 71:2622.
9Church. F. C.. G. L. Catignani, and H. E. Swaisgood. 1982. Use of immobilized Streptomyces griseus pro- teases (Pronase) as a probe of structural transitions of lysozyme, &lactoglobulin and casein. Enzyme Microb. Technol. 4:317.
locreamer, L. K. 1972. Hydrogen ion equilibria of bovine @-casein-B. Biochim. Biophys. Acta 271:252.
11 Creamer, L. K., T. Richardson, and D.A.D. Parry. 1981. Secondary structure of bovine all- and &wein in solution. Arch. Biochem. Biophys. 211:689.
12Creamer, L. K., H. F. Zoerb, N. F. Olson, and T. Richardson. 1981. Surface hydrophobicity of asl-l, aS1-casein A and B and its implications in cheese structure. J. Dairy Sci. 65:902.
13 Dalgleish, D. G. 1986. Analysis by fast protein liquid chromatography of variants of rtasein and their rele- vance to micellar structure and renneting. J. Dairy Res. 53~43.
14 Dalgleish, D. G., and T. G. Parker. 1980. Binding of calcium ions to bovine a,l-casein and precipitability of the proteincalcium ion complexes. J. Dairy Res. 47:113.
15Davies, D. T.. and A.J.R. Law. 1977. An improved method for the quantitative fractionation of casein mixtures using ion-exchange chromatography. J. Dairy Res. 44:213.
16 Dickson, I. R., and D. J. Perkins. 1971. Studies on the interactions between purified bovine caseins and alkaline-earth-metal ions. Biochem. J. 124:235.
17Dosak0, S.. T. Kimura, S. Taneya, T. Sone, S. Kaminogawa, and K. Yamauchi. 1980. Polymerization of a,l-casein by calcium ions. Agric. Biol. Chem. 44: 2443.
18 Eigel, W. N. 1977. Formation of yl-A2, y2-A* and y3- A caseins by in vitro proteolysis of &casein A2 with bovine plasmin. Int. J. Biochem. 8:187.
19 Eigel, W. N. 1981. Identification of proteose-peptone component 5 as a plasminderived fragment of bovine @-casein. Int. J. Biochem. 13:1081.
20Eige1, W. N., J. E. Butler, C. A. Ernstrom, H. M. Famll. Jr., V. R. Hanvalkar, R. Jenness, and R. McL. Whimey. 1984. Nomenclature of proteins of cow’s milk: fifth revision. J. Dairy Sci. 67:1599.
21 Farrell, H. M., Jr.. T. F. Kumosinski, P. Pulaski, and M. P. Thompson. 1988. Calcium-induced associations of the caseins: a thermodynamic linkage approach to precipitation and resolubilization. Arch. Biochem. Biophys. 265:146.
22Graham. E.R.B., G. N. Malcolm, and H. A. McKen- zie. 1984. On the isolation and conformation of bo- vine @-casein A. Int. J. Biol. Macromol. 6:155.
23 Ho, C., and D. F. Waugh. 1965. Interactions of bovine u,-casein with small ions. J. Am. Chem. Soc. 87:llO.
24 Ho, C., and D. F. Waugh. 1965. Interactions of bovine caseins with divalent cations. J. Am. Chem. SOC. 87: 889.
25 Hollar, C. M., A.J.R. Law, D. G. Dalgleish, and R. J. Brown. 1991. Separation of major casein fractions using cationexchange fast protein liquid chromatogra- phy. J. Dairy Sci. 742403.
26 Holt, C., and D.W.L. Hukins. 1991. Structural analysis of the enviionment of calcium ions in crystalline and amorphous calcium phosphates by x-ray absorption spectroscopy and a hypothesis concerning the biologi- cal function of the casein micelle. Int. Dairy J. 1:151.
27 Holt, C.. T. G. Parker, and D. G. Dalgleish. 1975. The thermochemistry of reactions between a,l-casein and calcium chloride. Biochim. Biophys. Acta 379:638.
28 Holt, C., and L. Sawyer. 1988. F’rimary and predicted secondary structures of the caseins in relation to their biological functions. Protein Eng. 2:251.
29Humphrey, R. S., and L. J. Newsome. 1984. High performance ion-exchange chromatography of the ma- jor bovine milk proteins. N.Z. J. Dairy Sci. Technol. 19:197.
tics of the interaction of calcium with casein sub- micelles as determined by analytical affinity chro- matography. Arch. Biochem. Biophys. 283:318.
30 Jang, H. D., and H. E. Swaisgmd. 1990. Chanlcteris-
Journal of Dairy Science Vol. 76, No. 10, 1993
SYMPOSIUM: GENETIC PERSPECTIVES ON MILK PROTEINS 306 1
31 Jenness, R., and J. Koops. 1962. Preparation and properties of a salt solution which simulates milk ultrafiltrate. Neth. Milk Dairy J. 16:153.
32Kaminogawa, S., K. Yamauchi, and C.-H. Yoon. 1980. Calcium insensitivity and other properties of asl-I casein. J. Dairy Sci. 63:223.
33 Kumosinski, T. F., E. M. Brown, and H. M. Farrell, Jr. 1991. Three-dimensional molecular modeling of bovine caseins: K-casein. J. Dauy Sci. 74:2879.
34Kumosinski, T. F., E. M. Brown, and H. M. Farrell, Jr. 1991. Three-dimensional molecular modeling of bovine caseins: a,l-casein. J. Dairy Sci. 74:2889.
35Loucheux-Lefebvre, M.-H., J.-P. Aube~t. and P. Jolles. 1978. Prediction of the conformation of the cow and sheep K-caseins. Biophys. J. 23:323.
36Loukas. S., D. Vamucha, C. Zioudrou, R. A. Streaty, and W. A. KIM. 1983. Opioid activities and structures of a-caseinderived exorphins. Biochemistry 22:4567.
37 Matoba, T., R. Hayashi, and T. Hata. 1970. Isolation of bitter peptides from tryptic hydrolysate of cssein and their chemical structure. Agric. Biol. Cbem. 34: 1235.
38 Meisel, H. 1986. Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein. Fed. Eur. Biol. Soc. Lett. 196:223.
39 Minamiura, N., Y. Matsumura, J. Fukumoto, and T. Yamamoto. 1972. Bitter peptides in cow milk casein digests with bacterial proteinase. Agric. Biol. Cbem. 36588.
40 Ng-Kwi-Hang, K. F., and J. P. Pelissier. 1989. Rapid separation of bovine caseins by mass ion exchange chromatography. J. Dauy Res. 56:391.
41 N o d i , Y., and C. Tanford. 1967. Examination of titration behavior. Methods Enzymol. 11:715.
420110, T., S. Kaminogawa, S. Odagiri, and K. Yamauchi. 1976. A study on the binding of calcium ions to a,l-casein. Agric. Biol. Chem. 4Q1717.
43 Ono, T., M. Yahagi, and S. Odagiri. 1980. The bind- ing of calcium to x-casein and para-K-casein. Agric. Biol. Chem. 44:1499.
44 Parker, T. G., and D. G. Dalgleish. 1981. Binding of calcium ions to bovine &casein. J. Dairy Res. 48:71.
45 Raap, J., K.E.T. Kerling, H. J. Vreeman, and S. Visser. 1983. Peptide substrates for chymosin (rennin): conformational studies of rc-casein and some n-casein- related oligopeptides by circular dichroism and secon- dary s t ructu~ prediction. Arch. Biochem. Biophys. 221:117.
46 Reimerdes, E. H.. and E. Herlitz. 1979. The formation of y-caseins during cooling of raw milk. J. Dairy Res. 46:219.
47 Ribadeau Dumas, 8. 1961. Fractionnement de la casCine par chromatographie sur colonne de diethyl-
aminoethyl-cellulose en milieu ude. Biochim. Bi- ophys. Acta 54:400.
48 Ribadeau Dumas, B., J. L. Maubois, G. Mocquot, and J. Gamier. 1964. Etude de la constitution de la caseine de vache par chromatographie sur colonnes de diethylaminoethyl-cellulose en milieu IN&. Biochim. Biophys. Acta 82:494.
49Shinoda, I., A. Fushimi, H. Kato, H. Okai, and S. Fukui. 1985. Bitkr taste of synthetic C-terminal tetradecapeptide of bovine &casein, H-Prol96-Val- Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile-Ile-Val*~- OH, and its related peptides. Agric. Biol Chem. 49: 2587.
50 Swaisgood, H. E. 1982. Chemistry of milk proteins. Page 1 in Developments in Dairy Chemistry-1 . P. F Fox, ed. Appl. Sci. Publ., London, Engl.
51 Swaisgood, H. E. 1992. Chemistry of caseins. Page 63 in Advanced Dairy Chemistry. Vol. 1. P F. Fox. ed. Elsevier Sci. Publ., Essex, Engl.
52 Swaisgood, H. E., and G. L. Catignani. 1987. Use of immobilized proteinases and peptidases to study struc- tutal changes in proteins. Methods Enzymol. 135596.
53 Reference deleted in proof. 54Takeuchi, M., E. Tsuda, M. Yoshikawa R. Sasak~.
and H. Chiba. 1985. Fractionation and characteriza- tion of 9 subcomponents of bovine r-casein A. Agric. Biol. Chem. 49:2269.
55 Thompson, M. P. 1966. DEAE-cellulose-urea chro- matography of casein in the presence of 2- mercaptoethanol. J. Dairy Sci. 49:792.
56 Toma, S. J., and S. Naikai. 1973. Calcium sensitivity and molecular weight of as5-casein. J. Dairy Sci. 56: 1559.
57van Dijk. H.J.M. 1990. The properties of casein micelles. 2. Formation and degradation of the micellar calcium phosphate. Neth. Milk Dairy J. 44: l l l .
58van Dijk. H.J.M. 1990. The properties of casein micelles. 3. Changes in the state of the micellar calcium phosphate and their effects on other changes in the casein micelles. Neth. Milk Dairy J. 44:125.
59 Vreeman, H. J., S. Visser, C. J. Slangen, and J.A.M van Riel. 1986. Characterization of bovine K-casein fractions and the kinetics of chymosin-induced macro- peptide release from carbohydrate-free and carbohydrate-containing fractions determined by high- performance gel-permeation chromatography. Bio- chem. J. 24087.
60 Yoshikawa, M., F. Tani, T. Yoshimura, and H. Chiba. 1986. Opioid peptides from milk proteins. Agric. Biol. Chem. 502419.
61 Yoshikawa, M., T. Yoshimura, and H. Chiba. 1984. Opioid peptides from human &casein. Agric. Biol. Chem. 48:3185.
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