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)