PROTEOLYSIS OF BOVINE ALPHA(S2)-CASEIN BY CHYMOSIN

Proteolysis of bovine α_s2-casein by chymosin (E. C. 3.4.23.4) in solution in 100 mm Na phosphate buffer, pH 6.5, at 30 °C was studied by reversed-phase (RP)-HPLC and urea-polyacrylamide gel electrophoresis (PAGE). Chymosin hydrolyzed α_s2-casein in solution to eight peptides detectable by urea-PAGE. Peptides soluble in acetate buffer, pH 4.6, were isolated by RP-HPLC on a C₁₈ column using an acetonitrile/water gradient and identified from their N-terminal amino acid sequence. The chymosin cleavage sites were at the bonds Phe₈₈-Tyr₈₉, Tyr₉₅-Leu₉₆, Gln₉₇-Tyr₉₈, Tyr₉₈-Leu₉₉, Phe₁₆₃-Leu₁₆₄, Phe₁₇₄-Ala₁₇₅ and Tyr₁₇₉-Leu₁₈₀. Chymosin cleavage sites were restricted to the hydrophobic regions of the molecule. The bond-type in α_s2-casein cleaved by chymosin was in agreement with that found to be susceptible to chymosin in other caseins. The primary site of chymosin action on α_s2-casein appeared to be at Phe₈₈-Tyr₈₉.