Search

The major component of caprine (goat) αs-casein has been isolated by DEAE- and CM-cellulose chromatography in buffers containing urea and 2-mercaptoethanol. The protein has a molecular weight of 25 700 as determined by gel filtration on Sepharose 6B in guanidine hydrochloride. Its composition, Asp17, Thr14, Ser14, Glu45, Pro18, Gly4, Ala10, Cys2, Val12, Met4, Ile12, Leu12, Tyr11, Phe8, His5, Lys22, Arg6, Trp2 and 7 phosphate residues, is much closer to that of bovine αs3-casein than to bovine αs1-casein. The caprine αs-casein is more easily precipitated with Ca2+ than bovine αs3-casein at 37°C, pH 6.8, which in turn is more easily precipitated than bovine αs1-casein.

The first major proteolytic cleavage during Cheddar cheese maturation is the scission of α s1 -casein to give a small peptide and α s1 -I. Previously this proteolytic step was correlated with changes in the rheological properties of cheese, notably a lower elasticity and a decrease in the force required to fracture the cheese. Using cis -parinaric acid and 1,8-anilinonaphthalene sulfonate, we showed that α s1 -I and α s1 -casein A have lower surface hydrophobicities than α s1 -casein B. The peptide segment involving residues 14 to 24 of the α s1 -casein B sequence must be important in the formation of a hydrophobic interaction site, and an extensive network of hydrophobically bonded α s1 -casein molecules is probably important in young cheese as well as in concentrated casein solutions.

Secondary-structure-prediction algorithms have been used to find the segments of beta-lactoglobulin sequence most likely to fit the circular dichroism assignment of 15% alpha-helix, 50% beta-sheet, and 15-20% reverse turn. A number of segments may have an alpha-helical conformation but the most prominent region of alpha-helix is from residue 129 to 143. A further probable alpha-helix segment is residues 65-76. The number of residues predicted to occur in segments of beta-sheet structure is less than expected. However, the most likely segments are for residues 1-6, 11-16, 39-45, 80-85, 92-96, 101-107, 117-123, and 145-151. Predicted reverse-turn tetrapeptides are residues 7-10, 49-52, 61-64, 88-91, and 112-115. These predicted secondary structures are consistent with the low-resolution structure of the molecule determined by X-ray diffraction studies.

High concentrations of high molecular weight peptides of low electrophoretic mobility occurred both in 11 mo old Gouda and 30 mo old Cheddar cheese. Three peptides isolated from each variety of cheese have been identified as γ -casein A 2 , TS-casein A 2 , and R-casein, by electrophoretic mobility, amino acid analysis, molecular weight determination, and mobility changes on gel electrophoresis in the presence of magnesium ions. The enzyme known as milk protease cleaves the β -casein A in milk between residues 28 and 29, 105 and 106, and 107 and 108 to give γ -, TS-, and R-caseins, respectively. It is probable, therefore, that β -casein is slowly degraded by milk protease in ripened cheese at a rate which is probably determined by the moisture content and pH of the cheese.

A conformational change at low pH in bovine β-lactoglobulin A has been studied by intrinsic fluorescence and fluorescence of the bound dye 8-anilinonaphthalene-1-sulphonate. Both studies show that when the pH of β-lactoglobulin solutions is altered between 6.5 and 2.0, a rapid change in protein conformation occurs, followed by a slower conformational change. It seems likely that the rapid changes are linked with the predominance of protein dimer at pH 6.5 and monomer at pH 2.0. The slow changes involve shifts in protein conformation of the region that includes one of the protein tryptophan residues.

Two pure caprine (goat) β-caseins were purified by DEAE- and CM-cellulose column chromatography in buffers containing urea and 2-mercaptoethanol. The molecular weight of each β-casein was found to be about 24 500 as determined by gel filtration on Sepharose 6B in guanidine·HCl, and each had the same amino acid composition; Asp9, Thr12, Ser15, Glu43, Pro33, Gly6, Ala5, Val21, Met6, Ile9, Leu20, Tyr4, Phe9, His5, Lys12, Arg3, Trp. Phosphate determinations showed, however, that β1-casein (the more mobile component on electrophoresis at pH 8.6) contained six residues per molecule, whilst β2-casein contained only five. This difference may have been caused by a gene duplication and mutation. Both caseins are similar to bovine β-casein in molecular weight and net charge.

All of the casein components could be adsorbed onto Octyl-Sepharose CL-4B from a phosphate buffer solution and separated chormatographically using a gradient of 0–6 M urea. β-Casein, despite its higher average hydrophobicity, was eluted first. The large peptides α s1 -I and β-I (derived from α s1 − and β-casein by chymosin action) were readily separated from their precurson proteins using this technique, and the rare A variant of α s1 -casein was well resolved from the commoner B variant.

Circular dichroism spectra of bovine α S1 - and β-casein in neutral buffers between 190 and 250 nm indicated that both proteins had low proportions of periodic structure, but more than in the denaturing solvents, 4.5 m guanidine-hydrochloride, or 6 m urea. The addition of low concentrations of calcium chloride to the protein solutions did not alter their spectra. Between 255 and 320 nm the spectrum of β-casein was the same in neutral buffer and in denaturing solvents indicating that the aromatic chromophores were in unordered segments of the sequence. The spectrum of α S1 -casein, however, increased in intensity in going from guanidine-hydrochloride to neutral buffer to buffered calcium chloride solution. Similar changes were apparent when carboxypeptidase A-treated α S1 -casein (which loses the C-terminal tryptophan residue) was substituted for α S1 -casein suggesting that the long-wavelength spectral changes arose from conformational changes involving the tripeptide tryptophan 164-tyrosine 165-tyrosine 166. A structure prediction program using primary sequence data indicated that segments 37–40, 50–69, 75–79, 95–100, and 116–128 of α S1 -casein may be helical while segments 22–25, 106–111, and 164–168 may be extended sheet structures. Similarly segments 2–5, 19–26, 42–45, and 92–106 of β-casein may be helical while segments 79–85, 155–158, 185–192, and 206–209 may be extended sheet structures. It is notable that the peptide bonds most sensitive to the milkcoagulating enzyme, chymosin (23–24 and 24–25 in α S1 -casein, and 189–190 and 192–193 in β-casein), are in short extended sheet structures surrounded by conformations predicted to be aperiodic or reverse turn.