Enzymochemical Studies on Snake Venoms. VII. Purification of Hemorrhagic Principle Ac4-Proteinase in the Venom of Agkistrodon acutus

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Abstract
Ac4-proteinase, one of proteinases of the venom of A. acutus, was purified by a combination of gel filtration on Sephadex G-75 and chromatography on CM-Sephadex C-50, 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 [to mice] and hemorrhagic [in rabbits] activities, but Ac4-proteinase had the only hemorrhagic activity. Hemorrhagic and proteolytic activities were inhibited by EDTA, Cys or antiserum but not by DFP, soybean trypsin inhibitor or chicken egg white trypsin inhibitor. The preparation was homogeneous as judged by disc electrophoresis over polyacrylamide gel at pH 8.3, sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focusing. The MW of this protein was approximately 33,000, and the isoelectric point was 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 .mu.g and 0.362 U/mg, respectively. This protein contained no carbohydrate.

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