Enzymochemical Studies on Snake Venoms. II. : Purification of Lethal Protein Ac1-Proteinase in the Venom of Agkistrodon acutus

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Abstract
A proteolytic enzyme named Ac1-proteinase was isolated from the venom of A. acutus by a combination of gel filtration on Sephadex G-75 and chromatographies on diethylaminoethyl (DEAE)-Sephadex A-50, DE32 cellulose and DEAE-Sephadex A-50. By these procedures, 38 mg of purified preparation was obtained from 1 g of crude venom. Ac1-proteinase also had lethal and hemorrhagic activities. These 3 activities were inhibited by EDTA or cysteine but not by soy bean trypsin inhibitor (SBTI) or diisopropyl fluorophosphate (DFP). The preparation was homogeneous as judged by disc electrophoresis at pH 8.3, and pH 7.5 polyacrylamide gels, immunodiffusion and isoelectric focusing. The MW of this protein was determined to be approximately 24,500 and the isoelectric point was pH 4.7 by isoelectric focusing with carrier ampholyte. The minimum hemorrhagic dose, LD50 and proteolytic activities of this protein were 0.223 and 76.5 .mu.g/mouse, and 0.544 unit, respectively. This protein did not contain any carbohydrates or nucleic acids.