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 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)
MILK PROTEINS 193 Arg(Variant B) H.Glu-GIn-Leu-Thr-Lys-Cys-Glu-Val-Phe -Glu-Leu-Lys-Asp-Leu-Lys-Gly-Tyr-Gly-Gly n(variant A al-Ser-Leu-Pro-Glu-Trp-Val-Cys-Thr-Thr-Phe-His-Thr-Ser-Gly-Tyr-Asp-Thr-Glu-Ala- lle-Val-GIn-Asn-Asn-Asp-Ser-Thr-Glu-Tyr-Gly-Leu-Phe-GIn-Ile-Asn-Asn-Lys-lle-Trp Cys-Lys-Asp-Asp-GIn-Asn-Pro-His-Ser-Ser-Asn-lle-Cys-Asn-lle-Ser-Cys-Asp-Lys-Phe- eu-Asp-Asp-Asp-Leu-Tht-Asp-Asp-lle-Met-cys- Val-Lys-Lys-lle-Leu-Asp-Lys-Val-Gly Ile-Asn-Tyr-Trp-Leu-Ala-His-Lys-Ala-Leu-Cys-Ser-Glu-Lys-Leu-Asp-GIn-Trp-Leu-Cys- nimo acad substitution s in genetic poly morphs rom brew and Groble 1992 Figure 4.25 Amino 4.8.4 and tertiary structure a-La is a compact globular protein, which exists in solution as a prolate ellipsoid with dimensions of 2.5 x 3.7 x 3.2 nm. It consists of 26% a-helix 14%B-structure and 60% unordered structure. The metal binding and molecular conformational properties of a-la were discussed in detail by Kronman (1989). The tertiary structure of a-la is very similar to that of lysozyme. It has been difficult to crystallize bovine a-la in a form suitable for X-ray crystallography but work on the detailed structure is at an advanced stage(Brew and Grobler, 1992) 4.8.5 Quaternary structure a-La associates under a variety of environmental conditions but the associ ation process has not been well studied 4.8.6 Other species a-La has been isolated from several species, including the cow, sheep, goat, sow, human, buffalo, rat and guinea-pig. Some minor interspecies differences in the amino acid sequence and properties have been reported. The milks of sea mammals contain very little or no a-la
MILK PROTEINS 193 1 I Are Y (Variant B) H. Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-F'he- -Glu-Leu-Lys- Asp-Leu-Lys-Gly-Tyr-Gl y-GlyGln (Variant A) 21 Val-Ser-Leu-Pro-Glu-Trp-Val-Cys-Thr-Thr-Phe-His-Thr-Ser-G1y-Tyr-Asp-Thr-Glu-AlaIle-Val-G1n-Asn-Asn-Asp-Ser-Thr-Glu-Tyr-Gly-Leu-Phe-Gln-Ile-Asn-Asn-Lys-Ile-TrpCy~-Lys-Asp-Asp-Gln-Asn-Pro-His-Ser-Asn-I~e-Cys-Asn-Ile-~er-Cys-Asp-Lys-P~e- 1 Leu-Asp-Asp-Asp-Leu-Thr-Asp-Asp-Ile-MetI -nrI Ile-Asn-Tyr-Trp-Leu-Ala-His-Lys-Ala-Leu-Cys-Ser-Glu-Lys-Leu-Asp-Gln-Trp-Leu-Cys- 121 123 Glu-Lys-Leu. OH I- Figure 4.25 Amino acid sequence of a-lactalbumin showing intramolecular disulphide bonds (-) and amino acid substitutions in genetic polymorphs (from Brew and Grobler, 1992). 4.8.4 Secondary and tertiary structure cc-La is a compact globular protein, which exists in solution as a prolate ellipsoid with dimensions of 2.5 x 3.7 x 3.2nm. It consists of 26% cc-helix, 14% p-structure and 60% unordered structure. The metal binding and molecular conformational properties of r-la were discussed in detail by Kronman (1989). The tertiary structure of a-la is very similar to that of lysozyme. It has been difficult to crystallize bovine a-la in a form suitable for X-ray crystallography but work on the detailed structure is at an advanced stage (Brew and Grobler, 1992). 4.8.5 Quaternary structure @-La associates under a variety of environmental conditions but the association process has not been well studied. 4.8.6 Other species a-La has been isolated from several species, including the cow, sheep, goat, sow, human, buffalo, rat and guinea-pig. Some minor interspecies differences in the amino acid sequence and properties have been reported. The milks of sea mammals contain very little or no a-la
DAIRY CHEMISTRY AND BIOCHEMISTRY One of the most interesting characteristics of a-lactalbumin is its role actos synthesis UDP-D-Galactose +D-glucose lactose UDP Lactose synthetase, the enzyme which catalyses the final step in the biosynthesis of lactose, consists of two dissimilar protein subunits, A and B: the A protein is UDP-galactosyl transferase while the B protein is a-la. In the absence of B protein, the a protein acts as a non-specific galactosyl transferase, i.e. it transfers galactose from UDP-galactose to a range of acceptors, but in the presence of B protein it becomes highly specific and transfers galactose only to glucose to form lactose(Km for glucose is reduced approximately 1000-fold ) a-Lactalbumin is, therefore, a'specifier protein and its action represents a unique form of molecular control in biological reactions. a-La from the milks of many species are effective modifier proteins for the UDP-galactosyl transferase of bovine lactose synthetase. How it exercises its control is not understood but it is suggested that the synthesis of lactose is controlled directly by z lactalbumin which, in turn, is under hormonal control(Brew and Grobler, 1992). The concentration of lactose in milk is directly related to the concentration of a-la; milks of marine mammals, which contain no x-la, contain no lactose. Since lactose is the principal constituent in milk affecting osmotic pressure, its synthesis must be controlled rigorously and this is the presumed physiological role of a-la Perhaps each molecule of a-la regulates lactose synthesis for a short period and is then discarded and replaced; while this is an expensive and wasteful se of an enzyme component, the rapid turnover affords a faster response should lactose synthesis need to be altered, as in mastitic infection, when the osmotic pressure of milk increases due to an influx of NaCl from the blood hapter 2) 4.8.8 Metal binding and heat stability a-La is a metallo- protein; it binds one Ca2+ per mole in a pocket containing four Asp residues( Figure 4.26); these residues are highly conserved in all az-la's and in lysozyme. The Ca-containing protein is quite heat stable (it is the most heat stable whey protein) or more correctly, the protein renatur llowing heat denaturation(denaturation does occur at relatively low temperatures, as indicated by differential scanning calorimetry). when the pH is reduced to below about 5, the Asp residues become protonated and lose their ability to bind Ca2+. The metal-free protein is denatured at quite low temperatures and does not renature on cooling; this characteristic has been exploited to isolate a-la from whey
194 DAIRY CHEMISTRY AND BIOCHEMISTRY 4.8.7 Biological function One of the most interesting characteristics of cc-lactalbumin is its role in lactose synthesis: UDP-D-Galactose + D-glucose ---+ lactose + UDP lactose synthetase Lactose synthetase, the enzyme which catalyses the final step in the biosynthesis of lactose, consists of two dissimilar protein subunits, A and B; the A protein is UDP-galactosyl transferase while the B protein is a-la. In the absence of B protein, the A protein acts as a non-specific galactosyl transferase, i.e. it transfers galactose from UDP-galactose to a range of acceptors, but in the presence of B protein it becomes highly specific and transfers galactose only to glucose to form lactose (K, for glucose is reduced approximately 1000-fold). cc-Lactalbumin is, therefore, a ‘specifier protein’ and its action represents a unique form of molecular control in biological reactions. cc-La from the milks of many species are effective modifier proteins for the UDP-galactosyl transferase of bovine lactose synthetase. How it exercises its control is not understood, but it is suggested that the synthesis of lactose is controlled directly by a-lactalbumin which, in turn, is under hormonal control (Brew and Grobler, 1992). The concentration of lactose in milk is directly related to the concentration of a-la; milks of marine mammals, which contain no x-la, contain no lactose. Since lactose is the principal constituent in milk affecting osmotic pressure, its synthesis must be controlled rigorously and this is the presumed physiological role of a-la. Perhaps each molecule of x-la regulates lactose synthesis for a short period and is then discarded and replaced; while this is an expensive and wasteful use of an enzyme component, the rapid turnover affords a faster response should lactose synthesis need to be altered, as in mastitic infection, when the osmotic pressure of milk increases due to an influx of NaCl from the blood (Chapter 2). 4.8.8 Metal binding and heat stability a-La is a metallo-protein; it binds one Ca2+ per mole in a pocket containing four Asp residues (Figure 4.26); these residues are highly conserved in all a-la’s and in lysozyme. The Ca-containing protein is quite heat stable (it is the most heat stable whey protein) or more correctly, the protein renatures following heat denaturation (denaturation does occur at relatively low temperatures, as indicated by differential scanning calorimetry). When the pH is reduced to below about 5, the Asp residues become protonated and lose their ability to bind Ca2+. The metal-free protein is denatured at quite low temperatures and does not renature on cooling; this characteristic has been exploited to isolate x-la from whey
MILK PROTEINS Figure 4.26 Calcium-binding loop in bovine a-lactalbumin(modified from Berliner et aL, 1991) 4.9 Blood serum albumin Normal bovine milk contains a low level of blood serum albumin(bSa) (0.1-0.4g1-1:0.3-1.0%of total N), presumably as a result of leakage from lood bsa is quite a large molecule(molecular mass c 66 kDa; 582 amino cids); its amino acid sequence is known. The molecules contain 17 disulphides and one sulphydryl. All the disulphides involve cysteines that are relatively close together in the polypeptide chain, which is therefore organ ized in a series of relatively short loops, some of which are shorter than others(Figure 4.27). The molecule is elliptical in shape and is divided into three domains blood, BSA serves various functions but it is probably of little significance in bovine milk, although it does bind metals and fatty acids; the latter characteristic may enable it to stimulate lipase activity. 4.10 Immunoglobulins (g) Mature milk contains 0.6-1 g Igl(c. 3% of total n) but colostrum contains up to 100", the level of which decreases rapidly postpartum (Figure 4.2)
MILK PROTEINS 195 Figure 4.26 Calcium-binding loop in bovine a-lactalbumin (modified from Berliner et a/., 1991). 4.9 Blood serum albumin Normal bovine milk contains a low level of blood serum albumin (BSA) (0.1-0.4gl-'; 0.3-1.0% of total N), presumably as a result of leakage from blood. BSA is quite a large molecule (molecular mass c. 66 kDa; 582 amino acids); its amino acid sequence is known. The molecules contain 17 disulphides and one sulphydryl. All the disulphides involve cysteines that are relatively close together in the polypeptide chain, which is therefore organized in a series of relatively short loops, some of which are shorter than others (Figure 4.27). The molecule is elliptical in shape and is divided into three domains. In blood, BSA serves various functions but it is probably of little significance in bovine milk, although it does bind metals and fatty acids; the latter characteristic may enable it to stimulate lipase activity. 4.10 Immunoglobulins (Ig) Mature milk contains 0.6-lg Igl-' (c. 3% of total N) but colostrum contains up to 1OOgl-', the level of which decreases rapidly postpartum (Figure 4.2)
196 DAIRY CHEMISTRY AND BIOCHEMISTRY 1 A Net charge 141A Figure 4.27 Model of the bovine serum albumin molecule Igs are very complex protei ich will not be reviewed here. Essential- ly, there are five classes of Ig IgG, IgD, IgE and IgM. IgA, IgG and gM are present in milk. These occur as subclasses, e.g. IgG occurs as IgG and IgG,. IgG consists of two long(heavy) and two shorter (light polypeptide chains linked by disulphides( Figure 4.28). IgA consists of two such units (i.e. eight chains) linked together by secretory component (SC) and a junction (J)component, while IgM consists of five linked four-chain units(Figure 4.29). The heavy and light chains are specific to each type of Ig. For a review of immunoglobuins in milk, see Larson(1992) The physiological function of Ig is to provide various types of immunity in the body. The principal Ig in bovine milk is IgG, while in human milk it is IgA. The calf (and the young of other ruminants)is born without Ig in its blood serum and hence is very susceptible to infection. However, the intestine of the calf is permeable to large molecules for about 3 days postpartum and therefore Ig is absorbed intact and active from its mothers milk; Igs from colostrum appear in the calves blood within about 3 h of
196 DAIRY CHEMISTRY AND BIOCHEMISTRY 41 0 141 A Net charge -10 -8 0 Figure 4.27 Model of the bovine serum albumin molecule. Igs are very complex proteins which will not be reviewed here. Essentially, there are five classes of Ig: IgA, IgG, IgD, IgE and IgM. IgA, IgG and IgM are present in milk. These occur as subclasses, e.g. IgG occurs as IgG, and IgG,. IgG consists of two long (heavy) and two shorter (light) polypeptide chains linked by disulphides (Figure 4.28). IgA consists of two such units (i.e. eight chains) linked together by secretory component (SC) and a junction (J) component, while IgM consists of five linked four-chain units (Figure 4.29). The heavy and light chains are specific to each type of Ig. For a review of immunoglobuins in milk, see Larson (1992). The physiological function of Ig is to provide various types of immunity in the body. The principal Ig in bovine milk is IgG, while in human milk it is IgA. The calf (and the young of other ruminants) is born without Ig in its blood serum and hence is very susceptible to infection. However, the intestine of the calf is permeable to large molecules for about 3 days postpartum and therefore Ig is absorbed intact and active from its mother’s milk; Igs from colostrum appear in the calves blood within about 3 h of
MILK PROTEIN ntigen binding s● c Figure 4.28 Model of the basic 7S immunoglobulin(Ig) molecule showing two heavy and two region: C, constant region: L, lig CHO, carbohydrate groups; Fab refers to the(top)antigen-specific portion of the Ig Fc refers to the cell-binding effector portion of the lg molecule(from Larson, 1992). kling and persist for about 3 months, although the calf is able synthesize its own Ig within about 2 weeks. It is, therefore, essential that a calf should receive colostrum within a few hours of birth. otherwise it will probably die. The human baby obtains Ig in utero and hence, unlike the calf, is not as dependent on Ig from milk (in fact its intestine is impermeable to g). However, the Ig in human colostrum is beneficial to the baby, e.g. it reduces the risk of intestinal infections As regards the type and function of Ig in colostrum, mammals fall into three groups(Figure 4.30)-those like the cow (i.e. other ruminants), those like the human, and some, e.g. the horse, with features of the other two groups(Larson, 1992)
MILK PROTEINS 197 Figure 4.28 Model of the basic 7s immunoglobulin (Ig) molecule showing two heavy and two light chains joined by disulphide bonds: V, variable region; C, constant region; L, light chain; H, heavy chain; 1, 2 and 3 subscripts refer to the three constant regions of the heavy chains; CHO, carbohydrate groups; Fab refers to the (top) antigen-specific portion of the Ig molecule; Fc refers to the cell-binding effector portion of the Ig molecule (from Larson, 1992). suckling and persist for about 3 months, although the calf is able to synthesize its own Ig within about 2 weeks. It is, therefore, essential that a calf should receive colostrum within a few hours of birth, otherwise it will probably die. The human baby obtains Ig in utero and hence, unlike the calf, is not as dependent on Ig from milk (in fact its intestine is impermeable to Ig). However, the Ig in human colostrum is beneficial to the baby, e.g. it reduces the risk of intestinal infections. As regards the type and function of Ig in colostrum, mammals fall into three groups (Figure 4.30) - those like the cow (i.e. other ruminants), those like the human, and some, e.g. the horse, with features of the other two groups (Larson, 1992)
