A new fluorogenic peptide substrate for vitamin K-dependent blood coagulation factor, bovine protein C

Protein C is a precursor of plasma serine proteinases, and its active form inactivates specifically blood coagulation Factor V and Factor VIII. Since a specific and sensitive synthetic substrate for the activated protein C was not known, we studied its amidolytic activity toward 25 fluorogenic peptides of the type peptidyl-4-methylcoumaryl-7-amide (peptidyl MCA). The activated protein C, namely, bovine protein C activated by bovine alpha-thrombin, showed the highest activity toward Boc-Leu-Ser-Thr-Arg-MCA. The enzyme's Km and Kcat values for this substrate were calculated to be 3.3 x 10(-4) M and 8.4 s-1, respectively. Optimum conditions for measurement of activated protein C activity were studied with this substrate. Optimum pH was 8.5. For the maximum activity at pH 8.5, concentrations of 0.1 M NaCl and 1 mM CaCl2 had to be maintained in the reaction mixture. The fluorogenic peptide Boc-Leu-Ser-Thr-Arg-MCA was successfully applied to a simple and accurate assay of protein C during its purification.