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Synthetic Peptides for Chymosin and Pepsin Assays: pH Effect and Pepsin Independent-Determination in Mixtures

Laboratoire de Biochimie Physique, INRA Service de Biologie Physicochimique, Université de Paris-Sud, 91405, ORSAY, France

ABSTRACT

Peptide I [H-Phe-Gly-His-Phe(NO₂)-Phe-Ala-Phe-OMe] hydrolyzed by chymosin with k_cat = .3 ± .3 s^-1 and K_M = 7 ± 3 mM (pH 4.7) inhibited competitively peptide II [H-Leu-Ser-Phe(NO₂)-Nle-Ala-Leu-OMe] hydrolysis by chymosin with K_I = .23 ± .12 mM at pH 4.7. In reference conditions (.4 mM peptide, .01 M acetate buffer pH 4.7), the specific activities of porcine pepsin and chymosin on peptide I were 470 ± 70 nM s^-1 and .8 nM s^-1 per mg of enzyme. This difference in specific activity for peptide I allowed development of a chymosin-independent pepsin assay for mixtures of these enzymes. In addition, peptide II with a specific activity of 2400 ± 300 nM s^-1 and 154 ± 20 nM s^-1 per mg of porcine pepsin and chymosin provides an alternative to measurement of milk clotting for measurement of chymosin- and pepsin-like activities in commercial rennets. Hydrolysis products of peptide II by chymosin exhibited one ionized group of apparent pK of 3.5 ± .2 and a molar absorption coefficient change of 1000 ± 100 at pH 4.7 and at 310 nm. From measurements of the kinetic constants, k_cat and K_M, from pH 2.5 to 7 with peptide II, chymosin activity depends on the protonation of one group of apparent pK 5.3 ± .2 in the free enzyme. Rennet powder proved to be fairly stable after a 17-month storage at 4 C. Within the same period, a crystalline chymosin solution kept at -18 C lost 30 to 50% of its activity.