BIOCHEMICAL-CHARACTERIZATION OF HEMORRHAGIC TOXIN FROM CROTALUS-VIRIDIS-VIRIDIS (PRAIRIE RATTLESNAKE) VENOM

Hemorrhage, necrosis and edema are some of the effects often observed following snake bites. This paper reports studies on the isolation and biological properties of hemorrhagic toxin from Crotalus viridis viri dis (Prairie rattlesnake) venom. A hemorrhagic toxin was isolated from C. v. viridis venom by Sephadex G-50, DEAE-Sephacel and Q-Sepharose c olumn chromatographies. The hemorrhagic toxin from C. v. viridis venom was shown to be homogenous as demonstrated by a single band on polyac rylamide gel electrophoresis and immunodiffusion. Its molecular weight was approximately 54,000 daltons, and it contained 471 amino acid res idues. The toxin possessed hemorrhagic activity with a minimum hemorrh agic dose (MHD) of 0.11 mu g and hydrolytic activity on dimethylcasein , casein, azocasein, azoalbumin, azocoll and hide powder azure. Hemorr hagic and casein hydrolytic activities were inhibited by EDTA, o-phena nthroline or dithiothreitol. The toxin contained 1 mole of zinc per mo le of protein and zinc is essential for both hemorrhagic and proteolyt ic activities. Hemorrhagic toxin possessed hydrolytic activity on the B-chain of insulin, which cleaves His(5)-Leu(6), His(10)-Leu(11), Ala( 14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25) bonds. This toxin als o hydrolyzed Ace and BP chains of fibrinogen. Intramuscular injections of hemorrhagic toxin caused an increase of creatine phosphokinase act ivity in mice serum from 50.3 mU/ml to 1133 mU/ml. A toxin isolated fr om C. v. viridis venom was shown to have strong hemorrhagic activity. Partial characterization is reported for this major hemorrhagic toxin in C. v. viridis venom.