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

432 BIOCHIMICA ET BIOPHYSICA ACTA BBA 35575 PURIFICATION AND SOME PROPERTIES OF HEMORRHAGIC PRINCIPLE r IN THE VENOM OF TRIMERESURUS FLA VOVIRIDIS* TAMOTSU OMORI-SATOH AND AKIRA OHSAKA 2nd Department of Bacteriology, National Institute of Health, Shinagawa-ku, Tokyo I4I (japan) (Received December 23rd, Ig6g) SUMMARY r. One of the hemorrhagic principles, hemorrhagic principle r, in the venom of Trimeresurusjlavoviridis was highly purified by gel filtration on Sephadex G-roo, by chromatography on DEAE-Sephadex A-so and on guanidoethyl cellulose and by recycling chromatography on Sephadex G-200. The hemorrhagic activity was de- termined quantitatively by the well-defined skin reaction and the parallel-line-as:ay. A 43-fold increase in specific activity was attained with a yield of zo% with respect to hemorrhagic activity. The minimum hemorrhagic dose of the purified preparation was o.oosS flg. 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. roo ooo (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 o.6% of the proteolytic activity present in the original ve