Characterization of a protein C activator from the venom of Agkistrodon contortrix contortrix

Abstract

An enzyme capable of activating protein C has been purified 60-fold from the venom of the Southern copperhead snake (Agkistrodon controtrix) by ion-exchange and gel filtration chromatography. The purified enzyme consists of a single polypeptide with an apparent molecular weight of 37000. The isoelectric point of the protein C activator was determined to be 6.3 when measured by chromatofocusing. The enzyme was inhibited by p-nitrophenyl p-guanidinobenzoate, phenylmethanesulfonyl fluoride, and D-Phe-Prp-Arg-Ch2Cl but was not affected by cysteine-directed reagents or by metal chelators. These results suggest that the enzyme is a serine protease. Protein C activator was capable of hydrolyzing the thrombin substraste tosyl-Gly-Pro-Arg-p-nitroanilide (TGPRpNA), and steady-state kinetic studies determined that the Km for amidolysis of this substrate was 1.1 mM while the Vmax was 66 s-1. The activator demonstrated considerable substrate specificity since the amidolysis of D-Phe-Pip-Arg-pNA, D-Ile-Pro-Arg-pNA, Bz-Ile-Glu-Gly-Arg-pNA, D-VAl-Leu-Arg-pNA, and pyrGlu-Pro-Arg-pNA was less than 10% of that of TGPRpNA when measured under identical conditions using 1.0 mM substrate concentrations. The enzyme appears to be thrombin-like in its preference for arginyl as compared to lysyl chloromethyl ketones as well as by its inhibition by benzamidine and p-aminobenzamidine. However, the substrate specificity of the activator is distinguished from .alpha.-thrombin in that it does not clot fibrinogen and does not react with antithrombin III or hirudin. The purified enzyme is an extremely effective activator of protein C, and gel studies demonstrated that activation of protein C was associated with cleavage of the heavy chain. Steady-state kinetic parameters revealed an apparent Km for protein C of 0.57 .mu.M and an apparent Vmax of 0.02 s-1. It was found that the activation of protein C was inhibited by CaCl2 and by NaCl. A Ki,app of 99 .mu.M and 120 mM was measured for the inhibition by Ca2+ and NaCl, respectively. Ca2+ and NaCl had no effect on the amidolysis of TGPRpNA catalyzed by the activator or on the inhibition of the enzyme by D-Phe-Pro-Arg-CH2Cl, suggesting that the effect of Ca2+ and NaCl likely results from their interaction with protein C.