Abstract
The caseins are the predominant class of proteins in the milk of all common dairying species. This class represents four phosphoproteins, i.e. αs1-, αs2-, β- and κ-casein. Their amino acid sequence combined with post-translational phosphorylation and, in the case of κ-casein, glycosylation yields a unique class of rheomorphic proteins which contain comparatively little secondary and tertiary structure and associate to form crucial building blocks for casein micelles. This chapter discusses the chemistry and biochemistry of caseins, with particular attention to primary and higher order structures, genetic variation, post-translational modification, disulphide interactions, self-association and interactions with minerals.
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4.1 Introduction
Milk protein constitutes an important part of the human diet. For the neonate, milk or infant formula is the only type of food consumed; however, whereas milk does not constitute a major part of the diet after the neonatal stage of most other mammals, the human diet in many parts of the world continues to include high levels of dairy products. The popularity of milk proteins in the human diet is undoubtedly a result of the combination of their excellent nutritional value and high level of functionality. The relative ease of isolation of proteins from milk has led not only to the creation of a wide variety of functional and nutritional milk protein ingredients, but also to milk proteins being the best characterized of all food proteins. The primary structures of all milk proteins have been determined and for all the major whey proteins, three-dimensional structures have been elucidated. Because of the fact that attempts to crystallize caseins have thus far remained unsuccessful, the full secondary and tertiary structure of the caseins remains to be elucidated. Although caseins have higher flexibility than typical globular proteins, e.g. whey proteins, the previous classification of caseins as random coil or natively denatured proteins appears inaccurate as a definite degree secondary and tertiary structure has been identified for the caseins.
The aim of this chapter is to provide the current state-of-the-art with respect to the chemistry of caseins. As with previous reviews on this topic by Swaisgood (1982, 1992, 2003), casein composition and nomenclature, chemical composition and primary structure of the caseins, post-translational modification, secondary structures as well as physicochemical properties of caseins, such as self-association and the interactions with calcium, will be covered. For studies on the isolation of casein, the reader is referred to Swaisgood (2003). Higher order structures of caseins and the structure and stability of the association colloids in which caseins naturally exist, i.e. casein micelles, are outside the scope of this chapter and are covered in Chaps. 5 and 6, respectively. The focus in this chapter will be on the caseins in bovine milk; interspecies variability in casein composition is covered in Chap. 13.
4.2 Casein Composition and Nomenclature
The American Dairy Science Association Committee on the Nomenclature, Classification and Methodology of Milk Proteins originally defined the bovine caseins as those phosphoproteins that precipitate from raw milk by acidification to pH 4.6 at 20°C (Jenness et al., 1956). Subsequent reports by the committee recommended that the caseins could be differentiated according to their relative electrophoretic mobility in alkaline polyacrylamide or starch gels containing urea, with or without β-mercaptoethanol (Whitney et al., 1976) or, more recently, according to their primary amino acid sequences (Eigel et al., 1984; Farrell Jr et al., 2004). Accordingly, four gene products can be identified: αs1-casein (αs1-CN), αs2-casein (αs2-CN), β-casein (β-CN) and κ-casein (κ-CN). Typical concentrations of αs1-CN, αs2-CN, β-CN and κ-CN in bovine milk are 12–15, 3–4, 9–11 and 2–4 g L-1, respectively, and the caseins account for ∼75–80% of total milk protein. For all caseins, various genetic variants have been identified. In addition, all caseins show considerable micro-heterogeneity, arising from post-translational modification; all caseins are phosphorylated, whereas glycosylation has been shown only for κ-CN. As discussed in further detail later, αs1-CN, αs2-CN and β-CN are classified as the calcium-sensitive caseins, whereas κ-CN is calcium insensitive. In addition to the aforementioned gene products, γ-caseins and λ-caseins have been identified, which arise from the hydrolysis of β-CN and αs1-CN, respectively, by the indigenous milk proteinase, plasmin. Enzymatic hydrolysis of milk proteins by plasmin is outside the scope of this chapter and is dealt with in detail in Chap. 12.
Current nomenclature of caseins and some casein fractions, as well as former classifications by which they were known, is shown in Table 4.1. In such nomenclature, a Latin letter indicates the generic variant of the proteins, whereas differences in the degree of post-translational modification are indicated by an Arabic number, followed by the letter P to indicate that the post-translational variation arises from phosphorylation. For example, αs1-CN B-8P refers to genetic variant B of αs1-CN containing eight phosphorylated amino acid residues. For each of the caseins, one of the variants outlined in Table 4.1 is considered to be the reference protein; these reference proteins are αs1-CN B-8P, αs2-CN A-11P, β-CN A2-5P and κ-CN A-1P.
4.3 αs1-Casein
4.3.1 Primary Structure of α s1-Casein
The αs1-CN family represents ∼40% of total casein in bovine milk. The reference protein for the αs1-CN family is αs1-CN B-8P, with ExPASy entry name and file number of CAS1_Bovin and P02662, respectively. The amino acid sequence of αs1-CN B-8P, which predominates in the milk of Bos taurus and was first established by Mercier et al. (1971) and Grosclaude et al. (1973), is shown in Fig. 4.1. The protein consists of 199 amino acid residues, with 8 of the 16 Ser residues in the protein being phosphorylated, i.e. Ser45, Ser47, Ser64, Ser66, Ser67, Ser68, Ser75 and Ser115 (Mercier et al., 1971). In αs1-CN B-9P, previously denoted αs0-CN, Ser41 is also phosphorylated (Manson et al., 1977). De Kruif and Holt (2003) identified two centres of phosphorylation in αs1-CN, i.e. f41–51, containing Ser41 (only in the 9P variant), Ser45 and Ser47, and f61-70, containing residues Ser64, Ser66, Ser67 and Ser68. These centres of phosphorylation are crucial in the stabilization of the calcium phosphate nanoclusters in the casein micelles (De Kruif and Holt, 2003).
The amino acid composition and properties of αs1-CN B-8P are shown in Table 4.2. Based on amino acid composition, the molecular mass of the protein prior to post-translational modification is estimated at ∼23.0 kDa, which increases to ∼23.6 kDa as a result of the phosphorylation of eight Ser residues. Based on the primary sequence, a pI of ∼4.9 would be expected for αs1-CN, but this decreases by ∼0.5 pH units through the phosphorylation of the eight Ser residues. Such values are in line with reported pI of αs1-CN varying from 4.4 to 4.8 (Trieu-Cuot and Gripon, 1981; Eigel et al., 1984). The aliphatic index, grand average hydropathicity (GRAVY) and hydrophobicity all suggest a moderately hydrophobic protein. αs1-CN B-8P contains 25 amino acid residues capable of carrying a positive charge and 40 capable of carrying a negative charge. A distribution of the charge over the polypeptide chain is shown in Fig. 4.2, which clearly highlights a positively charged N-terminus and a high concentration of negative charges, including the two clusters of phosphorylation, between residues 30 and 80. A moderate and even distribution of positive and negative charges is found between residues 81 and 150, whereas the remainder of the protein, with the exception of the 10 amino acid C-terminus, is largely unchanged. Distribution of hydrophobicity, according to the scale of Tanford (1962), of αs1-CN B is also shown in Fig. 4.2. In this scale, positive values represent a hydrophobic character whereas negative values represent a hydrophilic character. Some distinct patches of significant hydrophobicity can be observed, i.e. residues 20–35 and 160–175.
4.3.2 Genetic Variation of α s1-Casein
In addition to the B-variant of αs1-CN, a number of other genetic variants have been identified, an overview of which is shown in Table 4.3. In αs1-CN A, the amino acid residues 14–26 are missing as a result of exon skipping (Grosclaude et al., 1970); this variant has been found in Holstein Friesians, Red Holsteins and German Red cattle (Ng-Kwai-Hang et al., 1984; Grosclaude, 1988; Erhardt, 1993). Variant αs1-CN C predominates in the milk of Bos indicus and Bos grunniens (Eigel et al., 1984) and contains Gly instead of Glu at position 192 (Grosclaude et al., 1969). In αs1-CN D, which has been found in various breeds in France (Grosclaude, 1988) and Italy (Mariani and Russo, 1975) as well as in Jerseys in the Netherlands (Corradini, 1969), the Ala residue at position 53 is replaced by a phosphorylated Thr residue (Grosclaude et al., 1972). A replacement at position 59 of Gln by Lys and at position 192 of Glu by Gly yields αs1-CN E, which is found in Bos grunniens (Grosclaude et al., 1976), whereas αs1-CN F contains Leu instead of SerP at position 66 and is found in German Black and White cattle (Erhardt, 1993). Finally, αs1-CN G was discovered in Italian Brown cows (Mariani et al., 1995), but no amino acid sequence has been reported for this variant to date, whereas αs1-CN H arises from an eight amino acid deletion at positions 51–58 (Mahe et al., 1999).
4.3.3 Secondary Structure of α s1-Casein
The secondary structure of αs1-CN has been studied using a number of different approaches. While Fourier transform infrared (FTIR) spectroscopy studies by Byler and Susi (1986) found no secondary structure in αs1-CN, other studies have reported varying degrees of secondary structure elements in αs1-CN. The percentage of α-helix in αs1-CN has been estimated as 5–15% (Herskovits, 1966), 8–13% (Byler et al., 1988), 20% (Creamer et al., 1981) or 13–15% (Malin et al., 2005). For β-sheet, values of 17–20% were reported (Byler et al., 1988; Creamer et al., 1981), whereas Malin et al. (2005) reported 34–46% extended β-sheet-like structures in αs1-CN. In addition, 29–35% β-turn structures have been reported for αs1-CN (Byler et al., 1988). In addition, the presence of polyproline II structures in αs1-CN is evident from Raman optical activity spectra (Smyth et al., 2001). Higher order structures of caseins are described in further detail in Chap. 5.
4.3.4 Self Association of α s1-Casein
Self-association of αs1-CN is characterized by progressive strongly pH- and ionic strength-dependent consecutive self-association to dimers, tetramers, hexamers, etc. (Ho and Waugh, 1965; Payens and Schmidt, 1965, 1966; Schmidt and van Markwijk, 1968; Swaisgood and Timasheff, 1968; Schmidt, 1970a, b). At pH 6.6 and ionic strength >0.003, the monomers exist in a rapidly equilibrating equilibrium with oligomers; increasing ionic strength results in increasing association constants and the appearance of larger oligomers (Ho and Waugh, 1965; Schmidt and van Markwijk, 1968; Schmidt, 1970b). The free energy for formation of the various oligomers is comparable; hence, all species exist at appreciable concentrations, but they occur to different extents. At an ionic strength of 0.003, only monomers are present, whereas at an ionic strength of 0.01, a monomer–dimer equilibrium exists; at an ionic strength of 0.2, dimers and tetramers are favoured, while the formation of larger oligomers becomes progressively less favourable (Ho and Waugh, 1965; Schmidt, 1970b). Likewise, as the pH is increased above 6.6, the electrostatic repulsive free energy increases, resulting in smaller association constants yielding a lowered degree of association (Swaisgood and Timasheff, 1968). The larger association constants, and resulting much stronger association, of αs1-CN C compared to αs1-CN B can be explained by the change in electrostatic free energy (Schmidt, 1970a) due to its smaller net charge. However, αs1-CN D behaves identically to αs1-CN B (Schmidt, 1970a) although its net charge is greater than that of αs1-CN B. It should be noted that the αs1-CN B to αs1-CN D substitution, at position 53 (Table 4.3) occurs in the polar domain, whereas the αs1-CN B to αs1-CN C substitution, at position 192 (Table 4.3), occurs in the hydrophobic domain which is more likely to be in the association contact surface. Enzymatic deimination of five of the six Arg residues of αs1-CN reduces the susceptibility of the protein to self-association (Azuma et al., 1991).
4.3.5 Interactions of α s1-Casein with Calcium
When considering the interactions of αs1-CN, or any of the other caseins, with calcium, or other cations, two aspects should be considered, i.e. the binding of calcium by the protein and the calcium-induced precipitation of the protein by calcium. αs1-CN is one of the calcium-sensitive caseins; precipitation of αs1-CN occurs in the range of 3–8 mM CaCl2 (Schmidt, 1969; Bingham et al., 1972; Toma and Nakai, 1973; Dalgleish and Parker, 1980; Aoki et al., 1985; Farrell Jr et al., 1988) and occurs more readily for αs1-CN B than for αs1-CN A (Farrell Jr et al., 1988). When CaCl2 concentration exceeds ∼0.1 mM, the solubility of αs1-CN increases again, due to the salting-in effect (Farrell Jr et al., 1988). Calcium-induced precipitates of αs1-CN are readily solubilized in 4 M urea, suggesting that no calcium-induced cross-linkage of proteins occurred and that the driving forces behind the calcium-induced association are driven by hydrogen bonding and hydrophobic interactions in the absence of electrostatic repulsion (Aoki et al., 1985).
An extensive investigation into the calcium-binding and calcium-induced precipitation of αs1-CN by Dalgleish and Parker (1980) highlighted that the binding of calcium by the protein decreases considerably with increasing ionic strength. In addition, the concentration of calcium required to induce precipitation of αs1-CN also increases with increasing ionic strength, but not proportionally to calcium binding, i.e. the degree of calcium binding which is required to induce precipitation of αs1-CN decreases with increasing ionic strength (Dalgleish and Parker, 1980). Calcium binding by αs1-CN decreases when pH decreases below 7.0, but decreasing pH increases the concentration of calcium required to induce precipitation of αs1-CN (Dalgleish and Parker, 1980). Calcium-induced aggregation of αs1-CN was described as a monomer–octamer equilibrium, followed by Smoluchowski aggregation in which only the octamers participate (Dalgleish et al., 1981). Dephosphorylation reduces the number of calcium-binding sites on the protein and also reduces the stability of αs1-CN to calcium-induced precipitation (Yamuachi et al., 1967; Bingham et al., 1972; Aoki et al., 1985). Deimination of Arg residues in αs1-CN enhances calcium binding, as well as the stability of the protein to calcium-induced precipitation (Azuma et al., 1991).
Detailed analyses of the effects of calcium binding on αs1-CN have indicated several equilibria. The addition of up to 1 mM CaCl2 to αs1-CN induces an exothermic process, possibly hydrogen-bond formation (Holt et al., 1975), binding of calcium only to phosphorylated Ser residues (Ono et al., 1976) and the transfer of Tyr and Trp residues from an aqueous to an apolar environment (Ono et al., 1976). As the concentration of CaCl2 is increased from 1 to 3 mM, the aforementioned exothermic phase is followed by an increasingly endothermic reaction, possibly hydrophobic interactions (Holt et al., 1975); the burying of the aromatic chromophores is abated (Ono et al., 1976), whereas calcium binding by both phosphorylated Ser and carboxylate-containing residues occurs (Ono et al., 1976); turbidity increases slightly to a plateau level (Holt et al., 1975) and increasing numbers of bent-chain polymers are observed (Dosaka et al., 1980). Finally, between 3 and 5 mM calcium chloride, the reaction becomes very endothermic (Holt et al., 1975); binding of calcium, primarily to carboxylate-containing residues, continues (Ono et al., 1976); the turbidity increases dramatically (Holt et al., 1975) and precipitation eventually occurs. These results suggest that binding of Ca2+ to high-affinity phosphoseryl clusters in the polar domain alters its interaction with the hydrophobic domain, bringing about a conformational change in that domain which allows some association to occur. Further binding to carboxyl residues throughout the structure reduces the electrostatic repulsion and, consequently, interaction of the hydrophobic domains leads to the formation of large aggregates.
4.4 α s2-Casein
4.4.1 Primary Structure of α s2-casein
The αs2-CN family constitutes up to 10% of the total casein fraction in bovine milk and consists of two major and several minor components, and exhibits varying levels of phosphorylation (Swaisgood, 1992; Farrell Jr et al., 2009) and intermolecular disulfide bonding (Rasmussen et al., 1992, 1994). The reference protein for this family is αs2-CN A-11P, a single-chain polypeptide with an internal disulfide bond with ExPASy entry name and file number CAS2_Bovin and P02663, respectively. The primary structure of αs2-CN A-11P (Fig. 4.3), reported by Brignon et al. (1977), has been changed to Gln rather than Glu at position 87, as indicated by cDNA sequencing (Stewart et al., 1987) and DNA sequencing (Groenen et al., 1993). In addition to the aforementioned 11P variant of αs2-CN A, 10P, 12P and 13P forms of this protein have also been observed (Brignon et al., 1976). Three centres of phosphorylation have been identified, i.e. f8–16, which contains the phosphorylated residues Ser8, Ser9, Ser10 and Ser16; f56–63, which contains the phosphorylated residues Ser56, Ser57, Ser58 and Ser61; and f126–133, which contains the phosphorylated residues Ser129 and Ser131 (De Kruif and Holt, 2003).
The primary sequence of αs2-CN A-11P, as outlined in Fig. 4.3, contains two Cys residues, i.e. Cys36 and Cys40, which occur in intra- and intermolecular disulphide bonds. In αs2-CN isolated from bovine milk, >85% of the protein is in monomeric form containing the intramolecular disulphide bond, with the remaining fraction of αs2-CN consisting of dimers, which can be oriented parallel or antiparallel (Rasmussen et al., 1992, 1994). Brignon et al. (1977) pointed out that two very large segments of αs2-CN, of ∼80 residues, show very high sequence homology with each other and may arise from gene duplication. Sequence alignment by Farrell Jr et al. (2009) showed that the best homologous alignment was for residues 42–122 and 124–207. According to Farrell Jr et al., (2009), the αs2-CN molecule can be divided into five distinct regions. Residues 1–41 and 42–80 form typical casein phosphopeptide regions with high charge and low hydrophobicity, whereas residues 81–125 form a slightly positively charged region of high hydrophobicity and residues 126–170 form the so-called phosphopeptide analogue, with high negative charge but low phosphate content; finally, residues 171–207 have high positive charge and high hydrophobicity (Farrell Jr et al., 2009). Similar trends are available from the hydrophobicity and charge distribution in Fig. 4.4.
Some properties of αs2-CN are given in Table 4.4. The 207 amino acids yield a molecular mass of ∼24.3 kDa, which further increases to 25.2 kDa as a result of the phosphorylation of 11 Ser residues. For the non-phosphorylated polypeptide chain of αs2-CN, a pI of ∼8.3 is predicted, but the aforementioned phosphorylation of 11 Ser residues decreases pI considerably to ∼4.9. Because of the high level of charged residues, i.e. 33 residues able to carry a positive charge and 39 capable of carrying a negative charge, αs2-CN is generally regarded as the most hydrophilic of the caseins.
4.4.2 Genetic Polymorphism of α s2-Casein
The A variant of αs2-CN is most frequently observed in Western breeds. The B variant was observed with low frequencies in Zebu cattle in South Africa, but a specific site of mutation for αs2-CN B has not been identified to date. Variant αs2-CN C was observed in yaks in the Nepalese valley and the Republic of Mongolia (Grosclaude et al., 1976, 1982). As shown in Table 4.5, the C variant differs from the A variant at positions 33, 47 and 130, where Gly, Thr and Ile replace Glu, Ala and Thr, respectively (Mahe and Grosclaude, 1982). Variant αs2-CN D was observed in Vosgienne and Montbeliarde breeds (Grosclaude et al., 1978) and in three Spanish breeds (Osta et al., 1995). The D variant differs from αs2-CN A by the deletion of nine amino acid residues from positions 51–59 (Grosclaude et al., 1978), which is caused by the skipping of exon VIII, a 27-nucleotide sequence that encodes amino acid residues 51–59 (Bouniol et al., 1993).
4.4.3 Secondary Structure of α s2-Casein
Estimates of the secondary structure of αs2-CN have been obtained using a variety of techniques and show considerable differences. Garnier et al. (1978) suggested 54% α-helix, 15% β-sheet, 19% turns and 13% unspecified structure, whereas Hoagland et al. (2001) suggested 24–32% α-helix, 27–37% β-sheet, 24–31% turns and 9–22% unspecified structure. Furthermore, Tauzin et al. (2003) suggested 45% α-helix, 6% β-sheet and 49% unspecified structure, whereas 15% polyproline II structure was suggested by Adzhubei and Sernberg (1993). Most recently, Farrell et al. (2009) suggested 46% α-helix, 9% β-sheet, 12% turns, 7% polyproline II, 19% non-continuous α-helix or β-sheet and 7% unspecified secondary structure. Higher order structures of caseins are described in further detail in Chap. 5.
4.4.4 Association Properties of α s2-Casein
Given the aforementioned amphipathic and highly charged structure of αs2-CN, it is not surprising that its self-association properties strongly depend on ionic strength (Snoeren et al., 1980). αs2-CN associates less extensively than αs1-CN, but it does exhibit consecutive self-associations, the extent of which at 20°C reaches a maximum at an ionic strength of 0.2–0.3, but decreases at higher ionic strength (Snoeren et al., 1980). This perhaps unexpected decrease in association at higher ionic strengths may be due to ionic suppression of electrostatic interactions between the N-terminal and the C-terminal domains (Snoeren et al., 1980). Snoeren et al. (1980) assumed that αs2-CN particles under such conditions are spherical, which is indeed apparent from the electron micrographs reported by Thorn et al. (2008). However, when αs2-CN is incubated at higher temperatures, e.g. 37 or 50°C, ribbon-like fibrils with a diameter of ∼12 nm and length >1 μm, which occasionally form loop structures, are observed (Thorn et al., 2008). The formation of such fibrillar structures is optimal at pH 6.5–6.7 and more extensive at higher temperature. The presence of αs1-CN inhibits fibril formation by αs2-CN, whereas the presence of β-CN has little effect on αs2-CN fibril formation. Fibril formation is also reduced when the intra- and intermolecular disulphide bonds in αs2-CN are disrupted by the reducing agent, dithiothreitol (Thorn et al., 2008).
4.4.5 Interactions of α s2-Casein with Calcium
Of the caseins, αs2-casein has the highest number of phosphorylated residues and is also the most sensitive to calcium-induced precipitation. Calcium-induced precipitation of αs2-CN occurs at calcium concentrations less than 2 mM (Toma and Nakai, 1973; Aoki et al., 1985). As for αs1-CN precipitates, calcium-induced precipitates of αs2-CN are readily solubilized in 4 M urea, suggesting that no calcium-induced cross-linkage of proteins occurs and that the driving forces behind the calcium-induced interaction are driven by hydrogen bonding and hydrophobic interactions in the absence of electrostatic repulsion (Aoki et al., 1985). This is further substantiated by the fact that dephosphorylation of αs2-CN renders the protein insoluble at neutral pH, probably due to the low net charge on the protein at these conditions (Aoki et al., 1985; Table 4.4).
4.5 β-Casein
4.5.1 Primary Structure of β-Casein
The β-CN family constitutes up to 35% of the casein of bovine milk. The reference protein for this family, β-CN A2-5P, contains 209 residues and its ExPASy entry name and file number are CASB_Bovin and P02666, respectively. The protein was chemically sequenced by Ribadeau-Dumas et al. (1972), sequenced from its cDNA by Jimenez-Flores et al. (1987) and Stewart et al. (1987) and from its gene by Bonsing et al. (1988). The sequence for β-CN A2-5P is shown in Fig. 4.5. This sequence was corrected from the original sequences by Yan and Wold (1984) and Carles et al. (1988) and differs from the original sequences at four positions: Glu for Gln at positions 117, 175 and 195 and reversal of Pro137 and Leu138. The changes at residues 117 and 175 were confirmed by both groups and by gene sequencing, whereas the reversal of residues 137 and 138 is not in agreement with cDNA-sequencing data (Jimenez-Flores et al., 1987), which is in accordance with the original data. However, the Leu-Pro substitution is a one-base change, and mutations could occur and not be observed by HPLC-mass spectroscopy (MS) of peptides or by electrophoresis of the proteins. Preference is, however, given to the two aforementioned independent protein-sequencing reports. In a similar fashion, the change at position 195 is not in agreement with the cDNA results, but, in this case, three other lines of evidence support the occurrence of only Glu at residue 195, i.e. the two protein-sequencing corrections noted previously, the invariance on electrophoresis of β-CN (f108–209) from the A1, A2 and A3 genetic variants (Groves, 1969); and the purification from cheese of a bitter peptide β-CN (f193–209), the sequence of which is identical to the chemically corrected sequences (Gouldsworthy et al., 1996).
Some features of β-CN A2-5P are shown in Table 4.6, whereas the distribution of charge and hydrophobicity over the molecule is shown in Fig. 4.6. This 209 amino acid protein has a molecular mass which is increased from 23.6 kDa for the primary structure to 24.0 kDa following phosphorylation of the aforementioned five Ser residues. The pI of the non-phosphorylated amino acid is estimated at 5.1, which decreases to ∼4.7 as a result of phosphorylation, which is somewhat lower than experimental values of 4.8–5.0 observed by Trieu-Cuot and Gripon (1981). Some of the unique properties of β-CN are derived from the fact that it is strongly amphipathic. The N-terminus of β-CN, residues 1–40, contains essentially all the net charge of the molecule and has a low hydrophobicity and contains only two Pro residues. This section also contains the five phosphorylated Ser residues, i.e. Ser15, Ser17, Ser18, Ser19 and Ser35, of which the first four form a centre of phosphorylation (De Kruif and Holt, 2003). The middle section of β-CN, i.e. residues 41–135, contains little charge and moderate hydrophobicity, whereas the C-terminal, section 136–209, contains many of the apolar residues and is characterized by little charge and high hydrophobicity.
4.5.2 Genetic Polymorphism of β-Casein
In addition to the aforementioned A2 variant of β-CN, a number of other genetic variants have been observed. The amino acid substitutions giving rise to all variants of β-CN are given in Table 4.7. In addition, Chung et al. (1995) identified variant A4 in native Korean cattle using electrophoresis only; its substitutions compared to the A2 reference protein are thus far unknown. The A1 variant of β-CN differs from the A2 variant only by the substitution at position 67 of His for Pro (Bonsing et al., 1988), whereas the A3 variant contains Gln instead of His at position 106 (Ribadeau-Dumas et al., 1970). In addition, β-CN B contains the aforementioned mutation for the A1 variant, as well as Arg for Ser at position 122 (Grosclaude et al., 1974a). Likewise, β-CN C is also a variant of β-CN A1, which is not phosphorylated at Ser35 and contains Lys instead of Glu at position 37. β-CN D differs from β-CN A2 only at position 18, whereas it contains Lys instead of a phosphorylated Ser residue, whereas β-CN E contains Lys instead of Glu at position 36 (Grosclaude et al., 1974b). Visser et al. (1995) identified β-CN F, which contains the A1 substitution in addition to Leu for Pro at residue 152. Dong and Ng-Kwai-Hang (1998) identified β-CN G-5P, which is similar to β-CN A1 and F but contains a Leu in place of Pro at either position 137 or 138, depending on the sequence assigned, as the Pro-Leu reversal, as outlined above, is controversial. Han et al. (2000) showed that β-CN H1 represents two substitutions relative to the corrected reference β-CN A2, i.e. Arg to Cys at position 25 and Leu to Ile at position 88. A genetic variant, discovered by Senocq et al. (2002), was termed β-CN H2, which differs from the A2 variant at two known positions, i.e. Leu instead of Met at position 93 and Glu instead of Gln at position 72; in addition, a substitution of Gln to Glu occurs somewhere between residues 114 and 169 but was not located (Senocq et al., 2002). Finally, the I variant of β-CN was described by Jann et al. (2002) and contains only the Leu for Met substitution of the H2 variant at position 93.
4.5.3 Secondary Structure of β-Casein
Originally, β-CN was predicted to have little or no secondary structure and, with the exception of 10% α-helix, was predicted to occur as a random coil (Herskovits, 1966; Noelken and Reibstein, 1968), which was further supported by the results of Caessens et al. (1999). The presence of α-helix structure in β-CN was further shown by Creamer et al. (1981), Graham et al. (1984), Farrell Jr et al. (2001) and Qi et al. (2004, 2005), with values ranging from 7 to 25%. However, 15–33% β-sheet structure was also reported to be present in β-CN, as well as 20–30% turns (Creamer et al., 1981; Graham et al., 1984; Farrell Jr et al., 2001; Qi et al., 2004, 2005). Using optical rotary dispersion analysis, Garnier (1966) suggests that polyproline II could be an important feature in β-casein structure. Subsequent studies have indeed confirmed the presence of 20–25% polyproline II structure in β-CN (Farrell Jr et al., 2001; Syme et al., 2002; Qi et al., 2004). Higher order structures of caseins are dealt with in detail in Chap. 5.
4.5.4 Association Properties of β-Casein
The presence of distinct polar and hydrophobic domains in β-CN clearly manifests itself in the extremely temperature-dependent self-association behaviour of β-CN. At 0–4°C, primarily monomers of β-CN are observed (Payens and Van Markwijk, 1963), but even under these conditions, polymeric structure is not entirely absent (Farrell Jr et al., 2001). The hydrodynamic behaviour of β-CN under these conditions approaches that of a random coil, with the Stokes radius of 3.7 nm, determined by gel chromatography (Schmidt and Payens, 1972), agreeing well with values obtained by sedimentation and viscosity, and is also consistent with the 4–5-nm size of spherical particles observed by electron microscopy (Andrews et al., 1979). Small angle X-ray scattering indicates a radius of gyration of 4.6 nm (Schmidt and Payens, 1972, Andrews et al., 1979).
As the temperature is increased above 4–5°C, β-CN undergoes a highly cooperative, reversible, rapidly equilibrating discrete self-association, yielding large polymers with a narrow size distribution (Payens and Van Markwijk, 1963; Payens and Heremans, 1969; Payens et al., 1969; Schmidt and Payens, 1972; Niki et al., 1977; Andrews et al., 1979; Arima et al., 1979; Buchheim and Schmidt, 1979; Evans and Phillips, 1979; Takase et al., 1980; Schmidt, 1982; Thurn et al., 1987; Kajiwara et al., 1988; Leclerc and Calmettes, 1997a, b, 1998; Farrell Jr et al., 2001; De Kruif and Grinberg, 2002; O’Connell et al., 2003; Qi et al., 2004, 2005; Gagnard et al., 2007). The properties for this monomer–polymer equilibrium can be treated using a shell model for the polymer micelle with a continuous distribution of intermediates between the monomer and largest polymer micelle (Tai and Kegeles, 1984; De Kuif and Grinberg, 2002; O’Connell et al., 2003; Mikheeva et al., 2003). There appears to be a critical concentration above which micelles are formed, ranging from less than 0.5 mg/mL to about 2 mg/mL (Schmidt and Payens, 1972; Niki et al., 1977; Evans et al., 1979), which depends on the temperature, ionic strength and pH. The size of the polymer micelle has been characterized by the number of monomers in the polymer, estimates of which have been shown to vary from 15 to 60 (Schmidt and Payens, 1972; Buchheim and Schmidt, 1979; Takase et al., 1980; Thurn et al., 1987; Kajiwara et al., 1988; Farrell Jr et al., 2001); the radius of gyration, with varying estimates of 7.3–13.5 nm (Andrews et al., 1979; Thurn et al., 1987; Kajiwara et al., 1988); the Stokes radius of ∼15 nm (Niki et al., 1977; Thurn et al., 1987) and the radius observed by electron microscopy of 8–17 nm (Arima et al., 1979; Buchheim and Schmidt, 1979). Increasing ionic strength shifts the equilibrium towards the polymer micelle but affects the number of monomers in the micelle only slightly (Schmidt and Payens, 1972; Takase et al., 1980), whereas increasing the temperature shifts the equilibrium position and increases the number of monomers in the micelle (Takase et al., 1980). In the theoretical ratio, radius of gyration/Stokes radius is 0.775 for a hard sphere (Thurn et al., 1987; Kajiwara et al., 1988), while that observed for the β-CN polymer micelle is less than 0.6, suggesting the immobilization of water in a soft outer layer surrounding a more dense core (Kajiwara et al., 1988).
Removal of the C-terminal three hydrophobic residues, Ile-Ile-Val, greatly reduces the association (Thompson et al., 1967; Evans and Phillips, 1979), as does removal of the C-terminal 17 amino acids (Qi et al., 2005). Removal of these 17 amino acids (Qi et al., 2005) or the 20 C-terminal amino acids (Berry and Creamer, 1975) renders β-CN virtually incapable of binding the hydrophobic surface probe ANS. The importance of hydrophobic interactions in the micellization of β-CN is further exemplified by the enhanced micellization when H2O is replaced by D2O (Evans and Phillips, 1979) or when ethanol is added (Mikheeva et al., 2003) and by the reduced micellization of β-CN in the presence of urea (Mikheeva et al., 2003). The importance of charges on the N-terminus on the micellization of β-CN is strongly impaired by the absence of post-translational phosphorylation but this loss of micellization is partially restored by duplication of the 6 N-terminal amino acids of β-CN in expression (Gagnard et al., 2007).
4.5.5 Interactions of β-Casein with Calcium and Other Cations
Compared to αs1-CN and αs2-CN, β-CN is less sensitive to calcium-induced precipitation. At 37°C, β-CN precipitates in the range of 8–15 mM Ca2+ at 37°C (Schmidt, 1969; Parker and Dalgleish, 1981; Farrell Jr et al., 1988). However, at 1°C, β-CN remains in solution at concentrations up to 400 mM CaCl2 (Farrell Jr et al., 1988). Under physiological conditions, β-CN is capable of binding approximately seven calcium ions per molecule (Parker and Dalgleish, 1981; Baumy and Brule, 1988). Binding of calcium by β-CN increases with increasing temperature, whereas an increase in ionic strength reduces the binding of calcium by β-CN (Parker and Dalgleish, 1981; Baumy and Brule, 1988). In addition, the binding of calcium by β-CN decreases with decreasing pH (Baumy and Brule, 1988). The binding of other di- and trivalent cations has also been studied; binding of magnesium, zinc and manganese shows comparable dependence on pH and ionic strength to the binding of calcium, whereas the binding of iron and copper by β-CN is virtually independent of pH and ionic strength (Baumy and Brule, 1988). The amount of calcium required to induce precipitation of β-CN decreases strongly with increasing temperature, whereas decreases in the amount of calcium bound by β-CN at the point of precipitation are also observed (Parker and Dalgleish, 1981). Both dephosphorylation and glycation of β-casein have been shown to improve the stability of β-casein to calcium-induced precipitation (Darewicz et al., 1999).
4.6 κ-Casein
4.6.1 Primary Structure of κ-Casein
Within the caseins, κ-CN displays some rather unique features. It is the smallest of the caseins, is has a low level of phosphorylation, has a low sensitivity to calcium and is the only one of the caseins to occur in glycosylated form. The primary sequence of the 169 amino acid κ-CN A 1P, which is the parent protein of the κ-CN family and has the ExPASy entry name CASK_Bovin and file accession number P02668, is shown in Fig. 4.7. Like for the other caseins, variable degrees of phosphorylation have also been found for κ-CN. The monophosphorylated form of κ-CN appears to be phosphorylated exclusively at Ser149, whereas the diphosphorylated form of κ-CN is phosphorylated at Ser149 and Ser121 (Mercier, 1981; Minkiewicz et al., 1996; Talbo et al., 2001; Holland et al., 2006). For the triphosphorylated form of κ-CN, Holland et al. (2006) recently reported that the additional amino acid residue to be phosphorylated is not a Ser residue, but Thr145.
Some features of κ-CN A-1P are shown in Table 4.8, whereas the distribution of hydrophobicity and charge over the protein chain are shown in Fig. 4.8. Based on the amino acid sequence, it can be deduced that of the 169 amino acids, 17 can be positively charged, whereas 28 can be negatively charged and there are a further 14 aromatic residues. Both hydrophobicity and charge are distributed unevenly throughout the protein (Fig. 4.8). Negative charges are found only in the N-terminal fragment 1–20 and the C-terminal fragment 115–169; the intermittent fragment 21–114 is devoid of negatively charged residues. Additional negative charges arising from phosphorylation are also in the C-terminal segment 115–169, as would be negative charges arising from glycosylation, which, as discussed later, can occur on six Thr residues in this segment. Positive charges can be found in the N-terminal segment 1–116, but not in the C-terminal segment 117–169. Hydrophobicity distributions highlight, as for charges, an uneven distribution of hydrophobicity throughout κ-CN. Segment 1–20 shows predominantly hydrophilic behaviour, whereas segment 21–110 contains some strongly hydrophobic patches, which is in agreement with the absence of negatively charged and a low number of positively charged residues in this segment. Segment 110–120 is strongly hydrophilic, whereas the remainder, i.e. segment 121–169 shows some hydrophilic and hydrophobic areas. It should be noted that post-translational phosphorylation and glycosylation occurring in this part of the protein will reduce hydrophobicity considerably.
Not taking into account post-translational modification, the molecular mass of κ-CN A was reported as 19.0 kDa. Increases in mass arise from post-translational phosphorylation and glycosylation. Based on the amino acid sequence, a pI for κ-CN A of ∼5.9 can be expected. However, experimental observations have shown considerably lower values for the isoelectric point of κ-CN, as low as pH 3.5 (Holland et al., 2006), which is due to increased negative charges on the protein arising from post-translational phosphorylation and glycosylation. For the non-glycosylated monophosphorylated variants of κ-CN A and B, pI values of 5.56 and 5.81 were found by two-dimensional electrophoresis, with consistent reductions in pI apparent with increasing degree of phosphorylation and glycosylation (Holland et al., 2004).
4.6.2 Genetic Variation of κ-Casein
κ-CN A predominates in Western breeds, with the exception of Jerseys (Thompson and Farrell Jr, 1974; Bech and Kristiansen, 1990; Ng-Kwai-Hang and Grosclaude, 2003). In addition, a number of other variants of κ-CN have also been identified (Table 4.9). The major other variant of κ-CN is κ-CN B, which differs from κ-CN A by substitution at position 136 of Ile for Thr and at position 148 of Ala for Asp (Mercier et al., 1973). The C variant of κ-CN differs from κ-CN A by substitution of His for Arg at position 97 (Miranda et al., 1993). The E variant of κ-CN arises from a substitution at position 155, i.e. Gly for Ser (Miranda et al., 1993). κ-CN F1 was discovered in both Zebu and Black and White hybrid cattle and contains Val instead of Asp at position 148 (Sulimova et al., 1992). κ-CN F2 was reported to be a variant of κ-CN B, containing His instead of Arg at position ten (Prinzenberg et al., 1996). Erhardt et al. (1996) reported the occurrence of κ-CN G1 in alpine breeds, which, in addition to the substitutions occurring for κ-CN B, also contains Cys instead of Arg at position 97. κ-CN G2 was shown to occur in the milk of Bos grunniens and was shown to contain Ala instead of Asp at position 148. In Pinzgauer cattle, Prinzenberg et al. (1999) identified κ-CN H, which differed from κ-CN A by an Ile for Thr substitution at position 135. In another study, Prinzenberg et al. (1999) described κ-CN I, which differs from κ-CN A by Ala for Ser substitution at position 104. Finally, Mahe et al. (1999) described the occurrence of κ-CN J, which seems to have arisen from an Arg for Ser mutation at position 155 in Bos taurus cattle on the Ivory Coast. As outlined previously, however, κ-CN A and B predominate strongly in Western breeds of cattle.
From a technological perspective, the Phe105-Met106 bond in κ-CN is extremely important, as it is the hydrolysis of this bond by chymosin, or proteinases with comparable specificity, that initiates the gelation of milk, which will ultimately be processed into a cheese curd and a ripened or unripened cheese. The N-terminal segment 1–105 arising from the chymosin-induced hydrolysis of κ-CN is called para-κ-CN, whereas the C-terminal fragment 106–169 is called the caseinomacropeptide (CMP); when CMP is glycosylated, it is often referred to as glycomacropeptide (GMP). From Table 4.9, it is apparent that this sequence is conserved in all genetic variants of κ-CN. However, for κ-CN I, the adjoining Ser104 residue is replaced by the considerably more hydrophobic Ala residue. It is also worthwhile noticing that, as outlined further in later stages, all post-translational modifications of κ-CN occur in the CMP segment of the molecule.
4.6.3 Glycosylation of κ-Casein
Of the caseins, κ-CN is the only one for which post-translational glycosylation has been shown to occur. Vreeman et al. (1986) observed that ∼40% of κ-CN is non-glycosylated, whereas the remainder can contain up to six glycans. Glycosylation sites in κ-CN were found to be the Thr residues at positions 121, 131, 133, 142, 145 and 165 (Pisano et al., 1994; Molle and Leonil, 1995; Minkiewicz et al., 1996). Holland et al. (2004, 2005, 2006) showed that the different glycoforms of κ-CN can be separated readily by 2D electrophoresis on the basis of isoelectric point and molecular mass, yielding up to 16 different spots for κ-CN with isoelectric points down to ∼3.5. Such separations have laid the basis for the recent elucidation of the glycosylation pattern of κ-CN. Using tandem MS sequencing of chemically tagged peptides, it was observed that the mono-glycoform of κ-CN was glycosylated exclusively at Thr131, the di-glycoform exclusively at Thr131 and Thr142 and the tri-glycoform at Thr131, Thr133 and Thr142 (Holland et al., 2005). The tetra-glycoform of κ-CN B was shown to be glycosylated at Thr145, in addition to the three already-mentioned glycosylation sites, Thr131, Thr133 and Thr142 (Holland et al., 2006). The remaining two glycosylation sites of κ-CN were not confirmed by Holland et al. (2006) but are most likely, as proposed by Pisano et al. (1994) and Minkewicz et al. (1996), to be Thr121 and Thr165. In general, κ-CN B appears to be more heavily glycosylated than κ-CN A, also displaying a more complex and variable glycosylation pattern (Coolbear et al., 1996).
A variety of glycans have been shown to be attached to κ-CN, all of which have been shown to be attached to Thr residues. These glycans consist of galactose (Gal), N-acetylglucosamine (GalNAc) and N-acetyl neuraminic acid (NANA). The monosaccharide GalNac, the disaccharide Galβ(1–3)GalNac, the trisaccharides NANAcα(2–3)Galβ(1–3)GalNAc and Galβ(1–3)[NANAcα(2–6)]GalNac and the tetrasaccharide NANAcα(2–3)Galβ(1–3)[NANAcα(2–6)]GalNac have been identified attached to κ-CN. Saito and Itoh (1992) estimated the presence of 56.0% tetrasaccharide, 18.5% branched trisaccharide, 18.4% linear trisaccharide, 6.3% disaccharide and 0.8% monosaccharide.
4.6.4 Disulphide-Bonding Patterns of κ-Casein
The presence of the two Cys residues in κ-CN, i.e. Cys11 and Cys88, creates a complex disulphide-bonding pattern between κ-CN molecules in bovine milk. Swaisgood et al. (1964) showed that κ-CN obtained without reduction was apparently randomly cross-linked by intermolecular disulphide bonds, to give oligomers, with the smallest detectable oligomer having a mass of ∼60 kDa, corresponding to a trimer. The existence of disulphide-cross-linked oligomers has since been substantiated (Talbot and Waugh, 1970; Farrell Jr et al., 1988; Groves et al., 1992), with the further suggestion that, during biosynthesis, reduced monomers first interact with the calcium-sensitive caseins to form micelles, followed by random cross-linking by oxidation (Pepper and Farrell Jr, 1982). In κ-CN isolated from bovine milk, only ∼10% of total κ-CN appears to be in the monomeric form (Farrell Jr et al., 1996).
Both disulphide-cross-linked oligomers and reduced κ-CN are capable of forming polymer micelles and stabilizing calcium-sensitive caseins (Talbot and Waugh, 1970; Vreeman, 1979). In the monomeric form of κ-CN, Cys11 and Cys88 form an intramolecular disulphide bond. However, κ-CN complexes arising to octamers and larger have also been found in bovine milk. These complexes contain an apparently random distribution of disulphides, i.e. Cys11 to Cys88, Cys11 to Cys11, Cys88 to Cys11 and Cys88 to Cys88. Whether these patterns remain after isolation of the κ-CN from milk is strongly dependent on the physicochemical conditions of isolation. Particularly the presence of reducing agents such as β-mercaptoethanol and dithiothreitol will significantly impact the oligomeric distributions of κ-CN. As outlined in Table 4.9, κ-CN G1 even contains a third Cys residue, i.e. Cys97. The impact hereof on the disulphide-bonding pattern has, however, not been studied to date.
4.6.5 Secondary Structure of κ-Casein
The secondary structure of κ-CN has been studied using a number of methods. NMR studies by Rollema et al. (1988) suggest a high degree of flexibility, particularly in the macropeptide part of κ-CN. Some structure, however, has been detected for κ-CN using spectroscopic methods such as FTIR and CD. Estimates suggest that κ-CN may contain 10–20% α-helix, 20–30% β-structure and 15–25% turns (Byler and Susi, 1986; Griffin et al., 1986; Ono et al., 1987; Kumosinski et al., 1991, 1993; Sawyer and Holt, 1993; Farrell Jr et al., 1996, 2003). The degree of estimated α-helical structure in κ-CN increases with increasing temperature (10–70°C), while the proportion of β-structure and turns decreases with temperature (Farrell et al., 2003). In addition, analysis in the presence of alcohols also results in a higher degree of α-helix in κ-CN. Several structural motifs have also been suggested, including possible antiparallel and parallel β-sheets or βαβ structure in the hydrophobic domain (Raap et al., 1983) and a β-turn-β-strand-β-turn motif centred on the chymosin-sensitive Phe105-Met106 region (Creamer et al., 1998). The latter motif appears to be conserved in κ-CN from various species, as would be expected for specific sensitivity to aspartyl proteinases (Holt and Sawyer, 1988). Using a Raman optical activity study, Syme et al. (2002) identified the presence of polyproline II helical confirmation in κ-CN. Some of the predicted structure occurs in the polar macropeptide domain but the stability of ordered structure in a region of such high net charge and apparent hydration would seem questionable and contradicts the great deals of flexibility; this part of the molecule was found to exhibit in the NMR studies by Rollema et al. (1988).
4.6.6 Association Behavior of κ-Casein
When isolated from milk, κ-CN occurs in the form of multimeric complexes. Analysis by analytical ultracentrifugation suggests that the weight average molecular weight of these complexes is ∼1,180 kDa at 25°C and ∼1,550 kDa at 37°C (Groves et al., 1998). Electron microscopy studies have shown a radius of 5.0–7.5 nm, 9–10 nm (Parry and Carroll, 1969) or 8.9 nm (Farrell Jr et al., 1996). Similar values have been observed by gel permeation chromatography (9.4 nm; Pepper and Farrell Jr, 1982), dynamic light scattering (9.6 nm; Farrell Jr et al., 1996) and small angle neutron scattering (SANS), for which values for values of a radius of 7.4 nm (Thurn et al., 1987) and 8 nm (De Kruif et al., 2002) have been reported. Micelle size, structure and interaction radius were found to be independent of protein concentration (De Kruif et al., 2002). Both calcium and iron have been found to be present in isolated κ-CN, and their chelation by EDTA has been reported to result in disruption of the κ-CN particle, with subsequent aggregation into particles with a considerably broader size distribution (Farrell Jr et al., 1996).
Reduction of the disulphide bridges in aforementioned κ-CN particles leads to amphipathic monomers which can, like β-CN, associate into micellar structures; unlike the micellization of β-CN, micellization of reduced κ-CN shows no strong temperature dependence (Swaisgood et al., 1964; Vreeman et al., 1981). This suggests that micellization of reduced κ-CN is less dominated by hydrophobic interactions than micellization of β-CN. For the monomer–polymer micelle equilibrium of reduced κ-CN, the critical micelle concentration varies from 0.53 at an ionic strength of 0.1–0.24 mg/mL at an ionic strength of 1.0 (Vreeman, 1979; Vreeman et al., 1977, 1981). The degree of polymerization has been estimated at ∼30 κ-CN molecules per micelle, yielding a molecular mass of ∼570–600 kDa (Vreeman, 1979; Vreeman et al., 1981, 1986) and an estimated diameter of 23 nm (Vreeman et al., 1981). Such results are in agreement with values derived from SANS measurements on reduced κ-CN micelles; such measurements led De Kruif and May (1991) to conclude that reduced κ-CN micelles are spherical and consist of a dense core of ∼6–7 nm, surrounded by a more open outer layer, protruding up to 14.7 nm from the centre of the core. The interactions between micelles of reduced κ-CN can be described as that of the so-called hard spheres (De Kruif and May, 1991).
When reduced and carboxymethylated κ-CN was incubated at 37°C, it was observed in addition to spherical particles, there was also a high proportion of fibrillar structures present (Farrell Jr et al., 2003). The formation of such fibrillar structures, with a diameter of 10–12 nm and lengths up to 600 nm, was subsequently shown to occur for native, reduced and carboxymethylated κ-CN (Thorn et al., 2005; Ecroyd et al., 2008, 2010; Leonil et al., 2008). When native κ-CN is used, it is the dissociated form that is involved in fibril formation (Ecroyd et al., 2010). Fibril formation, which has been shown to result in an increased proportion of β-sheet structure (Ecroyd et al., 2008; Leonil et al., 2008), is more extensive at higher temperature (Thorn et al., 2005) and is more extensive for non-glycosylated κ-CN than for its glycosylated counterpart (Leonil et al., 2008). The presence of αs-CNs or β-CN inhibits fibril formation (Thorn et al., 2005; Leonil et al., 2008), whereas BSA does not inhibit fibril formation (Thorn et al., 2005). Segment Tyr25-Lys86 of κ-CN appears to be incorporated into the protease-resistant core of the fibrils (Ecroyd et al., 2008) whereas fragment 106–169, i.e., the macropeptide, in either glycosylated or non-glycosylated form, does not form fibrils under comparable circumstances (Leonil et al., 2008).
4.6.7 Interactions of κ-Casein with Calcium
Compared to the other caseins, interactions of calcium with κ-CN have studied far less. This is probably due to the fact that κ-CN is, unlike αs1-CN, αs2-CN and β-CN, the so-called calcium insensitive, i.e., it is not precipitated in the presence of excess calcium. Ono et al. (1980), studying the binding of calcium to κ-CN, observed that binding of calcium to phosphorylated Ser residues reached a plateau at 1 mM CaCl2, whereas binding of calcium by carboxyl groups increased linearly up to 3 mM CaCl2 and more slowly at higher concentrations. Spectra obtained from circular dichroism and UV analysis indicate that the binding of calcium to κ-CN does not induce changes in the secondary structure of the protein (Ono et al., 1980). Given the aforementioned potential role of calcium in stabilizing κ-CN particles isolated from milk (Farrell et al., 1996), further study on the interactions of calcium with κ-casein appears warranted.
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Huppertz, T. (2013). Chemistry of the Caseins. In: McSweeney, P., Fox, P. (eds) Advanced Dairy Chemistry. Springer, Boston, MA. https://doi.org/10.1007/978-1-4614-4714-6_4
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Print ISBN: 978-1-4614-4713-9
Online ISBN: 978-1-4614-4714-6
eBook Packages: Chemistry and Materials ScienceChemistry and Material Science (R0)