Purification and partial characterization of a new proteolytic enzyme fromthe venom of Bothrops moojeni (CAISSACA)

A basic serine protease which is active on casein and fibrinogen was purifi ed from Bothrops moojeni venom using a single step chromatography on a CM-S epharose fast flow column. The enzyme, MOO3, was not hemorrhagic and presen ted only a trace of blood-clotting activity. Synthetic chromogenic substrat es (azoacasein and azoalbumin) where not hydrolyzed by MOO3. Using polyacry lamide 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 mel. weight of 27,000, both i n the presence and absence of beta-mercaptoethanol. its pl was 7.8 by elect rofocusing. The enzyme did not contain neutral carbohydrates and its N-term inal amino acid was alanine. The amino acid composition showed 249 residues /mole, a high content of hydrophilic amino acids and 14 half-cystine residu es, which should account for 7 disulfide bonds. The protease cleaved the A- alpha chain faster than the B-beta of bovine fibrinogen and showed no effec t on the delta-chain. Specific esterolytic activity of MOO3 on alpha-N-tosy l-1-arginine methyl ester was 29.64 mu mol min(-1) x mg(-1). MOO3 represent ed 1.42% (w/w) of the initial desiccated venom. Its proteolytic activity wa s inhibited by beta-mercaptoethanol, leupeptin, phenyfmethylsulphonyI fluor ide and ethylenediamine tetraacetate.