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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.

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.