Purification and partial characterization of a new proteolytic enzyme from the venom of Bothrops moojeni (Caissaca)

Abstract

A basic serine protease which is active on casein and fibrinogen was purified from Bothrops moojeni venom using a single step chromatography on a CM-Sepharose fast flow column. The enzyme, MOO3, was not hemorrhagic and presented only a trace of blood-clotting activity. Synthetic chromogenic substrates (azoacasein and azoalbumin) where not hydrolyzed by MOO3. Using polyacrylamide gel electrophoresis at pH 4.3, MOO3 showed as a single protein band. Using sodium dodecyl sulfate-polyacrylamide electrophoresis, MOO3 behaved as a single-chain protein with an approximate mol. weight of 27,000, both in the presence and absence of β-mercaptoethanol. Its pI was 7.8 by electrofocusing. The enzyme did not contain neutral carbohydrates and its N-terminal amino acid was alanine. The amino acid composition showed 249 residues/mole, a high content of hydrophilic amino acids and 14 half-cystine residues, which should account for 7 disulfide bonds. The protease cleaved the A-α chain faster than the B-β of bovine fibrinogen and showed no effect on the δ-chain. Specific esterolytic activity of MOO3 on α-N-tosyl-1-arginine methyl ester was 29.64 μmol min-1 x mg-1. MOO3 represented 1.42% (w/w) of the initial desiccated venom. Its proteolytic activity was inhibited by β-mercaptoethanol, leupeptin, phenylmethylsulphonyl fluoride and ethylenediamine tetraacetate.