Abstract
Ac4-proteinase, one of proteinases of the venom of Agkistrodon acutus, was purified by a combination of gel filtration on Sephadex G-75 and chromatographies on carboxymethyl (CM) Sephadex C-50, diethylaminoethyl (DEAE) Sephacel, and DE52 Cellulose. By these procedures, 9.2 mg of purified preparation was obtained from 1 g of crude venom. Ac1-, Ac2- and Ac3-Proteinases obtained from the venom, possessed both lethal and hemorrhagic activities, but Ac4-proteinase had the only hemorrhagic activity. Hemorrhagic and proteolytic activities were inhibited by ethylenediaminetetraacetic acid (EDTA), Cysteine or anti-serum but not by diisopropyl fluorophosphate (DFP), soy bean trypsin inhibitor (SBTI) or chicken egg white trypsin inhibitor (EWTI). The preparation was homogeneous as judged by disc electrophoresis over polyacrylamide gel at pH 8.3, SDS polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weight of this protein was determined to be approximately 33000, and the isoelectric point was found to be pH 4.4 by isoelectric focusing with carrier ampholyte (pH 3-10). The minimum hemorrhagic dose and proteolytic activities of this protein were 0.307 μg and 0.362 unit/mg, respectively. This protein did not contain any carbohydrates.
