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Hypotensive and hemostatic properties of rattlesnake (Crotalus viridis helleri) venom and venom fractions in dogs
First American Symposium on Animal, Plant and Microbial Toxins
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direct fatal arrhythmogenic action of antharidin on the myocardium. The small but significant changes in hemoglobin and hematocrit probably result from splenic contraction of sympathetic origin . The mixed effect of antharidin on the tissue calcium concentrations is unexplained . Chemical modification of snake venom phosphollpaas A, : tryptophan residue modVication. PHILIP RosErtaERO', PEGGY BARRINGTON', ELEoNoRA CoNDREA', KAREN R. SooNs', CHEN-CHUNG YANG' ('Section of Pharmacology and Toxicology, The University of Connecticut, School of Pharmacy, Storrs, CT, U.S .A ., 'Rogoff-Wellcome Medial Research Institute, Tel-Aviv University, Medial School, Israel, and 'Institute of Molecular Biology, National Tsing Hua University, Hsinchu, Taiwan 300, Republic of China) . SNAKE venom phospholipases A, (PLA,) show aremarkable degree of amino acid sequence homology, yet differ markedly in enzymatic and pharmacological activities . For example, the basic PLA, from Nqja nigricollis (spitting cobra) venom has much greater lethal potency, ardiotoxicity, hemolytic and anticoagulant activity than the acidic PLA, from Ngja ngja atra (Taiwan cobra) venom, even though it has lower enzymatic activity than the acidic enzyme when tested on either natural or artificial substrates . In attempting to determine whether the same active site is responsible for both pharmacological and enzymatic activity, we have chemically blocked selective amino acid residues . Previous studies have shown that methylation or alkylation of histidine 48 at the enzymatic active site causes the loss of both enzymatic and pharmacological activities. In contrast carbamylation, acylation or guanidination of lysines causes a selective loss in pharmacological potency, while semiarbazide addition to the free carboxyl groups (aspartic andglutamic acids) causes, in some cases, a selective loss of enzymatic activity . Suggestions have been made that tryptophan (Trp) residues may be involved either in substrate binding at the enzymatic active site or in binding of the enzyme to lipid - water interfaces . We have therefore now extended our studies by modifying the tryptophan residues with 2-hydroxy-5-nitrobenzyl bromide (HNB-Br) . A derivative of N. n. atra PLA, having all three of its Trp residues modified showed 51 % and 24% of the native enzymatic activity when tested on lecithin -triton mixed micelles and on egg yolk, respectively . Derivatives of N. nigricollis PLA,, having 2.4 or 1.5 of its three Trp residues modified, had 55 - 70% of the native enzymatic activity when tested on lecithin - triton mixed micelles and egg yolk . Because of insolubility, the derivative having all three Trp residues modified could not be tested . The lethal potency of these derivatives was tested following intraventricular injection into the brains of rats and their electrophysiological cardiotoxic effects were monitored using intracellular recordings from the isolated ventricular wall of the rat heart. The decreases in lethal potency and cardiotoxicity were similar to the decreases noted above in enzymatic activity . Changes in anticoagulant and hemolytic potencies following Trp modification will also be presented. It is concluded that unblocked Trp residues are neither essential for enzymatic activity nor for pharmacological potency. Hypotensive and hemostatic properties of rattlesnake (Crotalus viridis helleri) venom and venom fractions in dogs. RICHARD C. SCHAEFFER JR, CAROLYN BRISTON, SHAWN-MARIE CHILTON and RICHARD W. CARLSON (Department of Medicine, Wayne State University, Detroit, MI, U.S .A .) . HYPOTENSIVE and hemostatic properties of southern Pacific rattlesnake (Crotalus viridis hellen) venom (100 Mg per kg, i .v . bolus) or venom fractions (40 fig per kg) were studied in mongrel dogs (n=27,15-27 kg). Venom was separated by gel filtration (Sephadex G-100) into three lethal fractions (FRI, 11 and III) . Non-reduced crude venom contained 8 main protein bands on SDS-10% polyacrylamide gel electrophoresis, with a molecular weight range of 115,000- 14,000 . FRI contained seven of these bands (115,000-14,000 mol. wt). A 30,000 mol. wt venom component represented 80% of FRII and a 19,000-14,000 mol. wt band comprised 94% of FRIII . Crude venom rapidly produced hypotension, thrombocyotpenia, hemolysis, the generation of fibrin monomers and a decrease in factor VIII :C activity . Fibrin/fibrinogen degradation products (FDP) appeared at + 15 min and fibrinogen concentration was significantly depressed at + 120 min. There was no significant change in prothrombin time or Factors 11, VII, X and XII activity. However, the partial thromboplastin time (PTT) was increased (P <0.05) by +30 min. Our data suggests that a high molecular weight thrombin-like venom component in FRI (>50,000 mol. wt) directly digests fibrinogen without activation of extrinsic or intrinsic coagulation factors . It appears likely that the thrombocytopenia is primarily the result of a c. 33,000 mol. wt platelet aggregating protein found in nearly equal amounts in FRI and FRIL A c. 30,000 mol. wt venom component in FRII was associated with hypotension and hemolysis. We show the appearance of fibrin monomers to be the first sign of thrombin-like venom-induced coagulopathy . Measurement of these products may provide more objective data to guide titration of antivenin therapy directly toward the early coagulation defect that occurs following rattlesnake venom poisoning in man. The fate of Crotalus atrox venom in the lung of the mouse: an electron microscopic immunochemical study. ROLF I . SCHIFF and JOSEPH F. GENNARO JR . (Department of Biology, New York University, New York, NY 10003, U.S .A .) . HORSERADISH peroxidase (HRP) was covalently conjugated to commercial antiserum against North American crotalid snake venoms and the conjugation product was tested in vitro for immunological specificity and
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direct fatal arrhythmogenic action of antharidin on the myocardium. The small but significant changes in hemoglobin and hematocrit probably result from splenic contraction of sympathetic origin . The mixed effect of antharidin on the tissue calcium concentrations is unexplained . Chemical modification of snake venom phosphollpaas A, : tryptophan residue modVication. PHILIP RosErtaERO', PEGGY BARRINGTON', ELEoNoRA CoNDREA', KAREN R. SooNs', CHEN-CHUNG YANG' ('Section of Pharmacology and Toxicology, The University of Connecticut, School of Pharmacy, Storrs, CT, U.S .A ., 'Rogoff-Wellcome Medial Research Institute, Tel-Aviv University, Medial School, Israel, and 'Institute of Molecular Biology, National Tsing Hua University, Hsinchu, Taiwan 300, Republic of China) . SNAKE venom phospholipases A, (PLA,) show aremarkable degree of amino acid sequence homology, yet differ markedly in enzymatic and pharmacological activities . For example, the basic PLA, from Nqja nigricollis (spitting cobra) venom has much greater lethal potency, ardiotoxicity, hemolytic and anticoagulant activity than the acidic PLA, from Ngja ngja atra (Taiwan cobra) venom, even though it has lower enzymatic activity than the acidic enzyme when tested on either natural or artificial substrates . In attempting to determine whether the same active site is responsible for both pharmacological and enzymatic activity, we have chemically blocked selective amino acid residues . Previous studies have shown that methylation or alkylation of histidine 48 at the enzymatic active site causes the loss of both enzymatic and pharmacological activities. In contrast carbamylation, acylation or guanidination of lysines causes a selective loss in pharmacological potency, while semiarbazide addition to the free carboxyl groups (aspartic andglutamic acids) causes, in some cases, a selective loss of enzymatic activity . Suggestions have been made that tryptophan (Trp) residues may be involved either in substrate binding at the enzymatic active site or in binding of the enzyme to lipid - water interfaces . We have therefore now extended our studies by modifying the tryptophan residues with 2-hydroxy-5-nitrobenzyl bromide (HNB-Br) . A derivative of N. n. atra PLA, having all three of its Trp residues modified showed 51 % and 24% of the native enzymatic activity when tested on lecithin -triton mixed micelles and on egg yolk, respectively . Derivatives of N. nigricollis PLA,, having 2.4 or 1.5 of its three Trp residues modified, had 55 - 70% of the native enzymatic activity when tested on lecithin - triton mixed micelles and egg yolk . Because of insolubility, the derivative having all three Trp residues modified could not be tested . The lethal potency of these derivatives was tested following intraventricular injection into the brains of rats and their electrophysiological cardiotoxic effects were monitored using intracellular recordings from the isolated ventricular wall of the rat heart. The decreases in lethal potency and cardiotoxicity were similar to the decreases noted above in enzymatic activity . Changes in anticoagulant and hemolytic potencies following Trp modification will also be presented. It is concluded that unblocked Trp residues are neither essential for enzymatic activity nor for pharmacological potency. Hypotensive and hemostatic properties of rattlesnake (Crotalus viridis helleri) venom and venom fractions in dogs. RICHARD C. SCHAEFFER JR, CAROLYN BRISTON, SHAWN-MARIE CHILTON and RICHARD W. CARLSON (Department of Medicine, Wayne State University, Detroit, MI, U.S .A .) . HYPOTENSIVE and hemostatic properties of southern Pacific rattlesnake (Crotalus viridis hellen) venom (100 Mg per kg, i .v . bolus) or venom fractions (40 fig per kg) were studied in mongrel dogs (n=27,15-27 kg). Venom was separated by gel filtration (Sephadex G-100) into three lethal fractions (FRI, 11 and III) . Non-reduced crude venom contained 8 main protein bands on SDS-10% polyacrylamide gel electrophoresis, with a molecular weight range of 115,000- 14,000 . FRI contained seven of these bands (115,000-14,000 mol. wt). A 30,000 mol. wt venom component represented 80% of FRII and a 19,000-14,000 mol. wt band comprised 94% of FRIII . Crude venom rapidly produced hypotension, thrombocyotpenia, hemolysis, the generation of fibrin monomers and a decrease in factor VIII :C activity . Fibrin/fibrinogen degradation products (FDP) appeared at + 15 min and fibrinogen concentration was significantly depressed at + 120 min. There was no significant change in prothrombin time or Factors 11, VII, X and XII activity. However, the partial thromboplastin time (PTT) was increased (P <0.05) by +30 min. Our data suggests that a high molecular weight thrombin-like venom component in FRI (>50,000 mol. wt) directly digests fibrinogen without activation of extrinsic or intrinsic coagulation factors . It appears likely that the thrombocytopenia is primarily the result of a c. 33,000 mol. wt platelet aggregating protein found in nearly equal amounts in FRI and FRIL A c. 30,000 mol. wt venom component in FRII was associated with hypotension and hemolysis. We show the appearance of fibrin monomers to be the first sign of thrombin-like venom-induced coagulopathy . Measurement of these products may provide more objective data to guide titration of antivenin therapy directly toward the early coagulation defect that occurs following rattlesnake venom poisoning in man. The fate of Crotalus atrox venom in the lung of the mouse: an electron microscopic immunochemical study. ROLF I . SCHIFF and JOSEPH F. GENNARO JR . (Department of Biology, New York University, New York, NY 10003, U.S .A .) . HORSERADISH peroxidase (HRP) was covalently conjugated to commercial antiserum against North American crotalid snake venoms and the conjugation product was tested in vitro for immunological specificity and