Enzymochemical Studies on Snake Venoms. IV. Purification of Lethal Protein Ac2-Proteinase in the Venom of Agkistrodon acutus

Abstract

Ac2-proteinase, 1 of proteinases of the venom of A. acutus was purified by gel filtration on Sephadex G-75, followed by chromatography on DEAE-Sephadex A-50 and DE52 cellulose. Purified preparation (12.8) 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. Ac2-proteinase possessed lethal and hemorrhagic activities. These activities along with proteolytic activities were inhibited by EDTA cysteine or antiserum but not by soybean trypsin inhibitor or DFP. The MW of Ac2-proteinase was estimated as about 25,000 by SDS[sodium dodecyl sulfate]-polyacrylamide gel electrophoresis and the isoelectric point was pH 4.9 by isoelectric focusing with carrier ampholyte. The minimum hemorrhagic dose, LD50, and proteolytic activities of the purified preparation were 0.431 .mu.g, 110 .mu.g/mouse and 0.173 unit/mg, respectively. This protein did not contain any carbohydrates or nucleic acids.