Purification and Some Properties of Hemorrhagic Principle I in the Venom of Trimeresurus Flavoviridis

Abstract

Summary 1. One of the hemorrhagic principles, hemorrhagic principle I, in the venom of Trimeresurus flavoviridis was highly purified by gel filtration on Sephadex G-100, by chromatography on DEAE-Sephadex A-50 and on guanidoethyl cellulose and by recycling chromatography on Sephadex G-200. The hemorrhagic activity was determined quantitatively by the well-defined skin reaction and the parallel-line-assay. A 43-fold increase in specific activity was attained with a yield of 20% with respect to hemorrhagic activity. The minimum hemorrhagic dose of the purified preparation was 0.0058 βg. Thus, the preparation possessed the highest hemorrhagic activity ever purified among the hemorrhagic principles of bacterial and animal origins. It also contained potent lethal toxicity (L.D.50 = 4.63 ftg per mouse). 2. The preparation was homogeneous as judged by electrophoresis on cellulose acetate membrane, isoelectric focusing and immunodiffusion but not homogeneous by ultracentrifugation. The hemorrhagic principle had a molecular weight of approx. 100 000 (s20,w = 5.8 S) and an isoelectric point of 4.3. The hemorrhagic activity was inhibited by such reagents as EDTA, cysteine or formaldehyde but not by DFP or soybean trypsin inhibitor. The preparation contained 7% of the lethal toxicity and 0.6% of the proteolytic activity present in the original venom. Possible relations of the hemorrhagic activity to the lethal toxicity and to the proteolytic activity are discussed.