THE ACTION OF CHYMOSIN ON KAPPA-CASEIN AND ITS MACROPEPTIDE - EFFECT OF PH AND ANALYSIS OF PRODUCTS OF SECONDARY HYDROLYSIS

Purified kappa-casein (A variant) and its macropeptide were subjected to hydrolysis by recombinant chymosin over the pH range 6.6-2.6. Total hydrolysates were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the 2% (w/v) trichloroacetic acid ( TCA)-soluble fraction by N-terminal sequencing and mass spectrometry f ollowing isolation of peptides by Mono S-and reverse-phase high perfor mance liquid chromatography (RP-HPLC). The maximum rate of hydrolysis of the Phe(105)Met(106) bond of kappa-casein was observed at pH 4.6. T he rate and extent of hydrolysis of the para-kappa-casein moiety incre ased with decreasing pH to a maximum at 3.6. At pH 6.6, the fragments Phe(18)-Leu(32), Ser(33)-Tyr(42), and Trp(76)-Phe(105) were detected a t low concentrations with one of the complementary fragments, Tyr(43)- Gln(75), being detected at pH 5.6 and below. These were the predominan t TCA-soluble secondary proteolytic products of kappa-casein at each p H, indicating preferential cleavage of the Phe(17)-Phe(18), Leu(32)-Se r(33), Tyr(42)-Tyr(43) and Gln(75)-Trp(76) bonds. Additional para-kapp a-casein cleavage sites were apparent, mostly at the lower pH values, these sites being PyroGlu(1)-Glu(2), Cys(11)-Glu(12), Tyr(30)-Val(31), Leu(50)-Ile(51), Trp(76)-Gln(77), Leu(79)-Ser(80), Asn(81)-Thr(82), S er(87)-Cys(88) and Met(95)-Ala(96). The macropeptide of rc-casein was relatively resistant to proteolysis at pH 6.6 and 5.6 but small C-term inal fragments, Thr(161)-Val(169), Val(162)-Val(169), Val(164)-Val(169 ) and Val(162)-Thr(167), were produced. At lower pH values, more exten sive hydrolysis of macropeptide occurred, with cleavage sites predomin antly located in the middle region of the molecule at the Thr(124)-Ile (125), Ala(126)-Ser(127), Glu(137)-Ala(138), Ala(138)-Val(139) and Ala (144)-Thr(145) bonds. (C) 1998 Elsevier Science Ltd. All rights reserv ed.