Previous page DAIRY CHEMISTRY AND BIOCHEMISTRY nd buffalo are roughly similar to those in bovine milk but those in human milk are very different, as will be discussed in section 4.13. B-Lg and a-la are synthesized in the mammary gland and are milk-specific; most of the other proteins in whey originate from blood or mammary tissue Since the 1930s, several methods have been developed for the isolation of homogeneous whey proteins, which have been crystallized (McKenzie, 1970, 1971). Today, homogeneous whey proteins are usually prepared 10n- exchange chromatography on Deae cellulose. 47阝 Lactoglobulin 4.7.1 Occurrence and microheterogeneity B-Lactoglobulin is a major protein in bovine milk, representing about 50% of total whey protein and 12% of the total protein of milk. It was among he first proteins to be crystallized, and since crystallizability was long considered to be a good criterion of homogeneity, B-lg, which is a typical globular protein, has been studied extensively and is very well characterized (reviewed by McKenzie, 1971; Hambling, McAlpine and Sawyer, 1992) Lg is the principal whey protein(WP)in bovine, ovine, caprine and buffalo milks, although there are slight interspecies differences. Some years ago, it was believed that B-lg occurred only in the milks of ruminants but it is now known that a closely related protein occurs in the milks of the sow, mare, kangaroo, dolphin, manatee and other species. However, B-lg does not occur in human, rat, mouse or guinea-pig milks, hich a-la is the principal WP. Four genetic variants of bovine B-1g, designated A, B, C and D, have been identified in bovine milk. A fifth variant, which contains carbohydrate, has een identified in the australian breed, Droughtmaster. Further variants occur in the milks of yak and Bali cattle Genetic polymorphism also occurs in ovine B-Ig 4.7.2 Amino acid composit The amino acid composition of some B-lg variants is shown in Table 4. 4 It is rich in sulphur amino acids which give it a high biological value of 110 It contains 2 moles of cystine and 1 mole of cysteine per monomer of 18 KD The cysteine is especially important since it reacts, following heat denatura- tion, with the disulphide of K-casein and significantly affects rennet coagu lation and the heat stability properties of milk; it is also responsible for the cooked favour of heated milk. Some B-lgs, e. g. porcine, do not contain a free sulphydryl group. The isoionic point of bovine B-lgs is c. pH 5.2
188 DAIRY CHEMISTRY AND BIOCHEMISTRY and buffalo are roughly similar to those in bovine milk but those in human milk are very different, as will be discussed in section 4.13. p-Lg and a-la are synthesized in the mammary gland and are milk-specific; most of the other proteins in whey originate from blood or mammary tissue. Since the 1930s, several methods have been developed for the isolation of homogeneous whey proteins, which have been crystallized (McKenzie, 1970, 1971). Today, homogeneous whey proteins are usually prepared by ionexchange chromatography on DEAE cellulose. 4.7 p-Lactoglobulin 4.7. I Occurrence and microheterogeneity P-Lactoglobulin is a major protein in bovine milk, representing about 50% of total whey protein and 12% of the total protein of milk. It was among the first proteins to be crystallized, and since crystallizability was long considered to be a good criterion of homogeneity, p-lg, which is a typical globular protein, has been studied extensively and is very well characterized (reviewed by McKenzie, 1971; Hambling, McAlpine and Sawyer, 1992). p-Lg is the principal whey protein (WP) in bovine, ovine, caprine and buffalo milks, although there are slight interspecies differences. Some years ago, it was believed that 13-lg occurred only in the milks of ruminants but it is now known that a closely related protein occurs in the milks of the sow, mare, kangaroo, dolphin, manatee and other species. However, p-lg does not occur in human, rat, mouse or guinea-pig milks, in which a-la is the principal WP. Four genetic variants of bovine p-lg, designated A, B, C and D, have been identified in bovine milk. A fifth variant, which contains carbohydrate, has been identified in the Australian breed, Droughtmaster. Further variants occur in the milks of yak and Bali cattle. Genetic polymorphism also occurs in ovine p-lg. 4.7.2 Amino acid composition The amino acid composition of some p-lg variants is shown in Table 4.4. It is rich in sulphur amino acids which give it a high biological value of 110. It contains 2 moles of cystine and 1 mole of cysteine per monomer of 18 kDa. The cysteine is especially important since it reacts, following heat denaturation, with the disulphide of Ic-casein and significantly affects rennet coagulation and the heat stability properties of milk; it is also responsible for the cooked flavour of heated milk. Some fl-lgs, e.g. porcine, do not contain a free sulphydryl group. The isoionic point of bovine p-lgs is c. pH 5.2. Previous Page
MILK PROTEINS 189 4.7.3 Primary structure The amino acid sequence of bovine B-lg, consisting of 162 residues per monomer is shown in Figure 4.22 4.7.4 Secondary structure B-Lg is a highly structured protein: optical rotary dispersion and circular dichroism measurements show that in the pH range 2-6, B-lg consists of 10-15% a-helix, 43%B-sheet and 47% unordered structure, includer 阝- turns 4.7.5 Tertiary structure The tertiary structure of B-lg has been studied in considerable detail using X-ray crystallography. It has a very compact globular structure in which the B-sheets occur in a B-barrel-type structure or calyx(Figure 4.23). Each monomer exists almost as a sphere with a diameter of about 3.6 nm H. Leu-lle-Val-Thr-GIn-Thr-Met-Lys-Gly-Leu-Asp-lle-GIn-Lys-Val-Ala-Gly-Thr-Trp-Tyr Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-lle-Ser-Leu-Leu-Asp-Ala-GIn-Ser-Ala-Pro-Leu-Arg Glu (variants A, B, c) GIn (variant A, BI Val-Tyr-Val-Glu. .Leu-Lys-Pro-Thr-Pro-Glu-Gly.Asp-Leu-Glu-lle-Leu-Leu .L His (Variant C) Asn-Glu-Cys-Ala-Gln-Lys-Lys-lle -lle-Ala-Glu. Lys-Thr-Lys-lle-Pro-ala- Val-Phe-Lys-lle-Asp-Ala-Leu-Asn-Glu-Asn-Lys-Val-Leu-Val-Leu-Asp-Thr-Asp-Tyr-Lys (Variant A) Val Lys-Tyr-Leu-Leu-Phe-Cys-Met-Glu-Asn-Ser-Ala-Glu-Pro-Glu-Gln-Ser-Le cys-GIn- Variant B, C) Ala 121 Cys-Leu-Vai-Ang-Thr-Pro-Glu-Val-Asp-Asp-Glu-Ala-Leu-Glu. Lys-Phe-Asp-Lys-Ala-Le Lys-Ala-Leu-Pro-Met-His-lle-Arg-Leu-Ser-Phe-Asn-Pro-Thr-GIn-Leu-Glu-Glu-Gln-Cys- Figure 4.22 Amino acid of bovine B-lactoglobulin, showing amino acid substit netic polymorphs and the intramolecular disulphide bonds (- )(from Swai
MILK PROTEINS 189 4.7.3 Primary structure The amino acid sequence of bovine p-lg, consisting of 162 residues per monomer, is shown in Figure 4.22. 4.7.4 Secondary structure p-Lg is a highly structured protein: optical rotary dispersion and circular dichroism measurements show that in the pH range 2-6, p-lg consists of 10- 15% a-helix, 43% P-sheet and 47% unordered structure, including p-turns. 4.7.5 Tertiary structure The tertiary structure of p-lg has been studied in considerable detail using X-ray crystallography. It has a very compact globular structure in which the ,&sheets occur in a p-barrel-type structure or calyx (Figure 4.23). Each monomer exists almost as a sphere with a diameter of about 3.6 nm. 1 H.Leu-Ile-Val-Thr-G1n-Thr-Met-Lys-Gly-Leu-Asp-Ile-Gln-Lys-Val-Ala-Gly-Thr-Trp-Tyr 21 Ser-Leu-Ala-Met-AIa-Ala-Ser-Asp-Ile-Ser-Leu-Leu-Asp-Ala-Gln-Ser-Ala-Pro-Leu-Arg 41 Glu (Variants A, B, C) Gln (Variant A,BI Val-Tyr-Val-Glu- -Leu-Lys-Pro-Thr-Pro-Glu-GIy-Asp-Leu-Glu-Ile-Leu-Leu- -LysGln (Variant D) His (Variant C) 61Wariant A) Asp I Trp-Glu-Asn- -Glu-Cys-Ala-G1n-Lys-Lys-Ile-Ile-Ala-Glu-Lys-Thr-Lys-I1e-Pro-Ala- (Variant B, C) Gly 81 Val-Phe-Lys-Ile-Asp-Ala-Leu-Asn-Glu-Asn-Lys-Val-Leu-VaI-Leu-Asp-Thr-Asp-Tyr-Lys- ................................................... 101 Lys-Tyr-Leu-Leu-Phe-Cjrs-Met-Glu-Asn-Ser-Ala-Glu-Pro-Glu-Gln-Ser-Leu- -Cys-Gln- (Variant A) Val ; SH (Variant B, C) Ala ...................... 15; SH Cys-Leu-Val-Arg-Thr-Pro-GIu-Val-Asp-Asp-Glu-Ala-Leu-Glu-Lys-Phe-Asp-Lys-Ala-Leu 141 Lys-Ala-Leu-Pro-Met-His-11e-Arg-Leu-Ser-Phe-Asn-Pro-Thr-Gln-Leu-GI-Glu-Gln-Cys- 161 162 His-Ile. OH L Figure 4.22 Amino acid sequence of bovine B-lactoglobulin, showing amino acid substitutions in genetic polymorphs and the intramolecular disulphide bonds (-, - - -) (from Swaisgood, 1982)
DAIRY CHEMISTRY AND BIOCHEMISTRY OoH Figure 4.23 Schematic representation of the tertiary structure of bovine B-lactoglobulin, howing the binding of retinol; arrows indicate antiparallel B-sheet structures (from Papiz et al,1986) 4.7.6 Quaternary structure by Timasheff and co-workers that below pH 3.5, B-1g dissociates to mono- ners of 18kDa. Between pH 5.5 and 7.5, all bovine B-lg variants form dimers of molecular mass 36 kDa but they do not form mixed dimers, i.e.a dimer consisting of A and B monomers, possibly because B-lg A and b contain valine and alanine, respectively, at position 178. Since valine is Larger than alanine, it is suggested that the size difference is sufficient to prevent the proper fit for hydrophobic interaction, Porcine and other B-Igs that contain no free thiol do not form dimers; lack of a thiol group is probably not directly responsible for the failure to dimerize Between pH 3.5 and 5. 2, especially at pH 4.6, bovine B-lg forms octamers of molecular mass 144 kDa. B-Lg A associates more strongly than B-lg B, possibly because it contains an additional aspartic acid instead of glycine (in B)per monomer; the additional Asp is capable of forming additional hydrogen bonds in the pH region where it is undissociated. B-Lg from Droughtmaster cattle, which has the same amino acid composition as bovine B-lg a but is a glycoprotein, fails to octamerize, presumably due to stearic hinderance by the carbohydrate moiety
190 DAIRY CHEMISTRY AND BIOCHEMISTRY Figure 4.23 Schematic representation of the tertiary structure of bovine /?-lactoglobulin, showing the binding of retinol; arrows indicate antiparallel 8-sheet structures (from Papiz et al., 1986). 4.7.6 Quaternary structure p-Lg shows interesting association characteristics. Early work indicated that the monomeric molecular mass of bovine 8-lg was 36 kDa but it was shown by Timasheff and co-workers that below pH 3.5, p-lg dissociates to monomers of 18kDa. Between pH 5.5 and 7.5, all bovine p-lg variants form dimers of molecular mass 36 kDa but they do not form mixed dimers, i.e. a dimer consisting of A and B monomers, possibly because p-lg A and B contain valine and alanine, respectively, at position 178. Since valine is larger than alanine, it is suggested that the size difference is sufficient to prevent the proper fit for hydrophobic interaction. Porcine and other p-lgs that contain no free thiol do not form dimers; lack of a thiol group is probably not directly responsible for the failure to dimerize. Between pH 3.5 and 5.2, especially at pH 4.6, bovine p-lg forms octamers of molecular mass 144kDa. p-Lg A associates more strongly than p-lg B, possibly because it contains an additional aspartic acid instead of glycine (in B) per monomer; the additional Asp is capable of forming additional hydrogen bonds in the pH region where it is undissociated. p-Lg from Droughtmaster cattle, which has the same amino acid composition as bovine p-lg A but is a glycoprotein, fails to octamerize, presumably due to stearic hinderance by the carbohydrate moiety
MILK PROTEINS Octamer H3555) Monomer Monomer (pH>7.5) Figure 4.24 Efect of ph on ernary structure of B-lactoglobulin Above pH 7.5, bovine B-lg undergoes a conformational change(referred to as the neR transition), dissociates to monomers and the thiol group becomes exposed and active and capable of sulphydryl-disulphide nter change. The association of B-lg is summarized in Figure 4.24 4.7.7 Physiological function Since the other principal whey proteins have a biological function, it has ong been felt that B-lg might have a biological role; it appears that this role lay be to act as a carrier for retinol(vitamin A). B-Lg can bind retinol in a hydrophobic pocket(see Figure 4.23), protect it from oxidation and transport it through the stomach to the small intestine where the retinol is transferred to a retinol-binding protein, which has a similar structure to B-lg B-Lg is capable of binding many hydrophobic molecules and hence its ability to bind retinol may be incidental. Unanswered questions are how retinol is transferred from the core of the fat globules, where it occurs in milk, to B-lg and how humans and rodents have evolved without B-lg
MILK PROTEINS 191 Octamer (pH3.5-5.5) 0 Monomer (pH < 3.5) Dimer (PH 5.5-7.5) I 0 Monomer (pH > 7.5) Figure 4.24 Effect of pH on the quaternary structure of 8-lactoglobulin. Above pH 7.5, bovine 13-lg undergoes a conformational change (referred to as the N PR transition), dissociates to monomers and the thiol group becomes exposed and active and capable of sulphydryl-disulphide interchange. The association of p-lg is summarized in Figure 4.24. 4.7.7 Physiological function Since the other principal whey proteins have a biological function, it has long been felt that p-lg might have a biological role; it appears that this role may be to act as a carrier for retinol (vitamin A). p-Lg can bind retinol in a hydrophobic pocket (see Figure 4.23), protect it from oxidation and transport it through the stomach to the small intestine where the retinol is transferred to a retinol-binding protein, which has a similar structure to p-lg. p-Lg is capable of binding many hydrophobic molecules and hence its ability to bind retinol may be incidental. Unanswered questions are how retinol is transferred from the core of the fat globules, where it occurs in milk, to p-lg and how humans and rodents have evolved without p-lg
DAIRY CHEMISTRY AND BIOCHEMISTRY B-Lg also binds free fatty acids and thus it stimulates lipolysis(lipases are inhibited by free fatty acids ); perhaps this is its physiological function. BSA also binds hydrophobic molecules, including fatty acids; perhaps BSa serves a similar function to B-lg in those species lacking B-lg 4.78 Denaturation Denaturation of whey proteins is of major technological significance and will be discussed in Chapter 9 4.8 a-Lactalbumin a-Lactalbumin(a-la)represents about 20% of the proteins of bovine whey (3.5% of total milk protein) it is the principal protein in human milk. It is a small protein with a molecular mass of c. 14 kDa. Recent reviews of the literature on this protein include Kronman( 1989) and Brew and Grobler (1992) 4.8.1Am The amino acid composition is shown in Table 4. 4. a-La is relatively rich in tryptophan(four residues per mole. It is also rich in sulphur(1.9%)which is present in cystine (four intramolecular disulphides per mole) and me- thionine; it contains no cysteine(sulphydryl groups). The principal -la's in no phosphorus or carbohydrate, although some minor forms may in either or both. The isoionic point is c. pH 4.8 and minimum solubility in 0.5 M NaCl is also at pH 4.8 4.8.2 The milk of Western cattle contains only z- la b but Zebu and Droughtmas- ter cattle secrete two variants, A and B a-La A contains no arginine, the one Arg residue of a-la b being replaced by glutamic acid 4.8.3 Primary structure The primary structure of a-la is shown in Figure 4.25. There is considerable homology between the sequence of a-la and lysozymes from many sources The primary structures of a-la and chicken egg white lysozyme are very similar. Out of a total of 123 residues in a la, 54 are identical to correspond ing residues in lysozyme and a further 23 residues are structurally similar (e.g. Ser/Thr, Asp/ Glu)
192 DAIRY CHEMISTRY AND BIOCHEMISTRY /I-Lg also binds free fatty acids and thus it stimulates lipolysis (lipases are inhibited by free fatty acids); perhaps this is its physiological function. BSA also binds hydrophobic molecules, including fatty acids; perhaps BSA serves a similar function to p-lg in those species lacking D-lg. 4.7.8 Denaturation Denaturation of whey proteins is of major technological significance and will be discussed in Chapter 9. 4.8 or-Lactalburnin a-Lactalbumin (a-la) represents about 20% of the proteins of bovine whey (3.5% of total milk protein); it is the principal protein in human milk. It is a small protein with a molecular mass of c. 14kDa. Recent reviews of the literature on this protein include Kronman (1989) and Brew and Grobler (1992). 4.8. I Amino acid composition The amino acid composition is shown in Table 4.4. a-La is relatively rich in tryptophan (four residues per mole). It is also rich in sulphur (1.9?40) which is present in cystine (four intramolecular disulphides per mole) and methionine; it contains no cysteine (sulphydryl groups). The principal a-la’s contain no phosphorus or carbohydrate, although some minor forms may contain either or both. The isoionic point is c. pH 4.8 and minimum solubility in 0.5 M NaCl is also at pH 4.8. 4.8.2 Genetic variants The milk of Western cattle contains only r-la B but Zebu and Droughtmaster cattle secrete two variants, A and B. a-La A contains no arginine, the one Arg residue of a-la B being replaced by glutamic acid. 4.8.3 Primary structure The primary structure of a-la is shown in Figure 4.25. There is considerable homology between the sequence of a-la and lysozymes from many sources. The primary structures of r-la and chicken egg white lysozyme are very similar. Out of a total of 123 residues in r-la, 54 are identical to corresponding residues in lysozyme and a further 23 residues are structurally similar (e.g. Ser/Thr, Asp/Glu)