Batroxobin is a thrombin-like enzyme purified from Bothrops atrox moojeni venom. The enzyme hydrolyzed arginine esters, such as benzoyl-L-arginine ethyl ester (Bz-Arg-OEt) and tosyl-L-arginine methyl ester (Tos-Arg-OMe), but scarcely hydrolyzed tosyl-L-lysine methyl ester (Tos-Lys-OMe) and benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt). Batroxobin also hydrolyzed arginine amides, such as benzoyl-L-arginine-4-methyl-coumaryl-7-amide (Bz-Arg-MCA) and benzoyl-L-arginine-p-nitroanilide (Bz-Arg-pNA) but the rates were slower than the arginine esters. Optimal pH for hydrolysis of Bz-Arg-MCA by batroxobin was 8. The enzyme was relatively heat-resistant at pH 3 to 7 but not at alkaline pH. Bz-Arg-MCA hydrolytic activity of batroxobin was inhibited by diisopropyl fluorophosphate (DFP), antipain, leupeptin and benzamidine. Trypsin inhibitors, such as N
2-p-tosyl-L-lysine chloromethyl ketone (TLCK), soybean trypsin inhibitor, Trasylol and α
1-antitrypsin, and specific thrombin inhibitors, such as antithrombin III and hirudin were ineffective. The inhibition constant (K
i) for antipain, leupeptin and benzamidine were 0.12μM, 4.7μM, 0.58 mM, respectively.
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