Enzymochemical Studies on Snake Venoms. V. Purification of Lethal Protein Ac3-Proteinase in the Venom of Agkistrodon acutus

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Abstract
Ac3-Proteinase was purified from the lyophilized venom of A. acutus using gel filtration on a Sephadex G-75 column, followed by chromatography on DEAE-Sephadex A-50, DEAE-Sephacel and DEAE-Sephacel rechromatography. By these procedures, 22.7 mg of purified preparation was obtained from 1 g of crude venom. The purified preparation was homogeneous as judged by disc electrophoresis over polyacrylamide gel at pH 8.3 and isoelectric focusing. Ac3-Proteinase possessed both lethal and hemorrhagic activities. These and proteolytic activities were inhibited by EDTA, Cys or antiserum, but not by soybean trypsin inhibitor (SBTI), p-chloromercuribenzoate or DFP. The MW of Ac3-proteinase was estimated to be about 57,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was found to be pH 4.7, indicating that the protein was acidic. The minimum hemorrhagic dose, LD50 and proteolytic activities of the purified preparation were 0.95 .mu.g, 108 .mu.g/mouse, and 0.253 unit/mg, respectively. Both Ac1- and Ac2-proteinases were simple proteins, while Ac3-proteinase was a glycoprotein.