FRACTIONATION OF AGKISTRODON BILINEATUS VENOM BY ION EXCHANGE CHROMATOGRAPHY

Abstract

The enzyme activities and their pH optima for crude lyophilized Agkistrodon bilineatus venom were as follows: phosphomonoesterase (pH 9.5), phosphodiesterase (pH 9.0), 5'-nucleotidase (pH 10.0 to 10.2), phospholipase A, thrombin-like, N-benzoyl-L-arginine ethyl esterase (pH 8.0), p-toluene-sulphonyl-L-arginine methyl esterase (pH 8.0), protease (pH 8.9 to 9.1), and L-amino acid oxidase (pH 7.5 to 8.5). The crude venom was fractionated into 19 components by disc electrophoresis and into four distinct molecular weight groups by using thin layer gel filtration with Sephadex G-100 and G-200 superfine. Separation by DEAE Sephadex A-50 with ammonium acetate buffer by two stage elution yielded 13 fractions; 9 occurred in the first stage elution and four in the second stage. Eluants constituting the 13 fractions obtained by ion exchange chromatography had the following distribution of enzyme peak activities: phosphomonoesterase (Fractions IX and X), phosphodiesterase (I and III), 5'-nucleotidase (III and IX), phospholipase A (V and XI), esterase between Fractions IX and X, protease (X), L-amino acid oxidase (IV and VI). Enzyme activities in the eluants of the fractions were many times greater than the activities observed in the crude venom. The peaks of enzyme activities usually did not coincide with the major peaks of the protein fractions. Using the fractions obtained by ion exchange chromatography, phospholipase A enzymes were purified.