Abstract
One of the arginine ester hydrolases (TAME hydrolases) of Trimeresurus mucrosquamatus venom was isolated by a combination of gel filtration on Sephadex G-100, ion exchange chromatographies on CM-Sephadex C-50, DEAE-Sephacel and chromatofocusing on PBE 94. From 2 g of the venom 10.1 mg of purified TAME hydrolase, ME-4 was obtained. The substrate specificity of ME-4 was strictly directed to the hydrolysis of arginine ester. The esterolytic activity was inhibited by benzamidine, p-tosyl-L-phenylalanine chloromethyl keton (TPCK) or diisopropyl fluorophosphate (DFP).
ME-4 was proved to be homogeneous by electrophoresis on polyacrylamide gel and isoelectric focusing. The molecular weight was found to be about 28, 500. Its isoelectric point was 5.31. This enzyme was a glycoprotein. The esterolytic activity of the final preparation was 152.5 units/mg (substrate; TAME). This enzyme had capillary permeability-increasing and kinin-releasing activities, but not clotting activity. This protein was stable to heat treatment, and between pH 4 and 12. Its Michaelis constant (Km) for TAME and inhibition constants (Ki) for benzamidine, DFP and TPCK were found to be 25.0×10-3 M, 0.63×10-3 M, 4.44×10-3 M and 0.57×10-3 M, respectively.
