Rattlesnake venom poisoning and hemostatic defects

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found to be reversible both for fluoroalcohols and for SDS unfolding. The reversibility of the unfolding-refolding process of cardiotoxin in aqueous mixtures of fluoroalwhols was dependent on the volume per volume ratio of alcohol to water. SDS did not unfold the sernndary structures of cardiotoxin, whereas its tertiary structure was affected . If the SDS concentration is aqueous solution exceeded the critical micelle concentration value of SDS, a quasi-refolded state of cardiotoxin was observed . The mechanism of unfolding-refolding is discussed in terms of molecular interactions which might govern the protein conformation in solution (Authors abstract) C. C. Ywwc Ywtvc, C. C., Huwr~o, C. S. and Lee, H. J. (Institute of Molecular Biology, National Taing Hua University, Hsinchu, Taiwan 300, R.O .C .) Studies on the status of tyroayl residues in phoapholipesea A= from Ngja ngja afro and Ngja nigricoUis snake venons. J. Protein Chem . 4, 87 (1985). PH06PHOLIPASeS A, (PLA,) from Ngjo ngJa afro and Ngja nigrirnUis snake venons were subjected to tyrosine modification with p-nitrobenzenesulfonyl fluoride (NBSF) at pH 8.0. Three major NBS derivatives from each PLA, were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence . Spectrophotometric titration indicated that the phenolic groups of Tyr-3 and Tyr~3 (or Tyrfi2) had pK values of 10 .1 and 11 .0, respectively . The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca'', however, the reactivity of Tyr-63 (or Tyr~2) toward NHSF was greatly enhanced by Ca" . Modifiation of Tyr~3 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity . Conversely, modification of Tyr-3 in N. ngja afro PLA= couldcause more than a six-fold increase in lethal potency, in sharp contrast to the loss of enzymatic activity. Tyrosine-63-modified N. ngja afro PLA, exhibited the same Ca''-induced difference spoors as that of native PLA,, indicting that the Ca"-binding ability of Tyr-63-modifed N. ngja star PLA, was not impaired. However, Tyr-3-modified PLA, and all Tyr-modified N. ntgricolUs CMS-9 wen sot perturbed by Ca=', revealing that the Ca='-binding ability have boalost after tyrosine modifiction . These results suggest that Tyr_ 62 in N. nigrloollis CMSA and Tyr-3 is both enzymes are involved in Ca" binding. At pH 8.0 both active PLA, enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonaie (ANS) dramatically, whik all of the Tyr-modified derivatives did not enhance theemission intensity at all, elther in the presence or absence of Ca=', suggesting that the hydrophobk pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr~3 (or Tyr-62) an involved . (Authors abstract) C. C. YwNa TWO

Y~r~c, C. C. and CIiANO, L. S. (Institute of Molecular Biology, National Tang Hun University, Hsinchu, Taiwan 300, R.O.C .) Tryptophan modification of pho:pholipase A, enzymes and preaynaptic neurotoxins from snake venons . J. Protein Chem . 3, 195 (1984) . Txrtee pho:pholipasea A1 (PLA~ purified from cobra venons and two presynaptically acting neurotoxins that exhibit PLA3 activity were subjected to tryptophan modification with 2-hydroxy-S-nitrobenryl bromide. Assocâated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whams antigenicity remained unchanged . The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was conmstent with the relative reactivity toward 2-hydroxy-5nitrobenzyl bromide, except for Xemarhatus Iwentachanrs PLA=, which showed unusually high reactivity due to its characteristic dimeric conformation . Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and S00 nm, with a maximum of 41 S nm. Scatchard plots revealed only one type of binding rite for Ca'' , and thebinding abilities of the modified enzymes were not impaired. At pH 8.0 all native enzymes enhanced the emission intensity of 8anilinonaphthalene aulfonate (ANS) dramatically and the emission intensity of the ANS-enzyme complex increased or deceased in parallel with increasing concentration of Ca=' for the respective enzyme . The Trpmadified derivatives did not enhance the emission iatmaity of ANS at all, dther in the presence or absence of Ca". By means of tryptophan modification we wa~e able to infer that the tryptophan residues are in the vicinity of the Ca" binding site and are directly involved is the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the PLA, enzyme with the substrate, suggests that the Trp residues in PLA, enzymes and preaynaptic toxins are involved in substrate binding . C. C. Y~na (Authors abaract) Coatua~, J. J., Jerert, M. and Rus~t.i, F. E. (Department of Pediatric and Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ 83721, U.S.A.) . Rattlesnake venom poisoning and hemostatic defects. T7tromb. HarmoJtas. S4, 314 (1983) .

Heatosr~nc defects arc common following the bites of most rattlesnakes . These may be due to vascular injury, thrombocytopeaia, adisseminated intravaacular coagulation-like (DIC) syndrome and/or hyperflbrinolyais. The

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vrnoma of four rattlesnakes were analyzed for fibrinogen clotting activity, ability to induce platelet aggregation and Sbrinolytic activity. The results are shown in the Table below . Vrnom

FPL "

PAt

CLOT

Crotales molosses molasses Crotales atrox Crotales adamantees Crotales stele/alas scetebtes

4 .25 S .SO 0 .23 0 .25

0 .15 0 .073 None None

None None Positive None

"Fibrin plan lyais (urokinase equivalrnt units/mg vrnom) tPlaulet aggregation (least concentration, mg/ml, for >80Si aggregation) C.m . molasses and C. atrox venoma displayed high fibrinolytic activity, whereas those of C. adomarrtees and C.s. scetrrlates were weak . Cm . molasses and C. atrox vrnoms also caused platelet aggregation, and only C. adamantees had a dotting principle. The studies are consistent with clinical observations on bias by these rattlesnakes . In grneral, bites by C.s. scetulatus do not cause a coagulopathy, whereas C. atrox and C.m . molasses bites can provoke a significant defibrination syndrome secondary to the flbrinolytic activity . This will be demonstrated in clinical cases . Russet,t., F. E . (Dcpartmrnt of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721, U .S .A .) Snake venons . Symp. zool. Soc. Lond., No . 52, 469 (1984) . SNAxE vrnoms are complex mixtures, chiefly proteins, many of which have enzymatic properties . Some of the more lethal proteins of snake vrnoms are peptides, while other oomponrnts are metalloproteina, glycoprotelns, lipids, biogenic amines, free amino adds and inorganic substances, such as sodium, calcium, potassium, magnesium, zinc and other metals. The primary funcxion of a snake vrnom is to immobilize and, in moat cases, kill the prey . A snood function relates to the digestion of the prey and a third property implicates its use in a defensive posture against predators . The discharge of a snake vrnom as a 'marking' or `tagging' agent for the location of food following rnvrnomation seems questionable . In studying venom it is common to use techniques that fractionate the poison into individual parts . Thus, a destructive praoeas is used to study what evolved as a constructive process . The consequence is that the function of the whole venom is sometimes lost sight of in the preoccupation with the individual parts. This has led to labeling wrnoms (or even vrnom componrnts) with such misleading terms as 'neurotoxins', 'cardiotoxins', 'hemotoxins', 'myotoxina' and the like, obscuring the evolution of the important synergistic, metabolic and sutopharmacological phenomena of the whole vrnom . The function of a snake venom is dosdy interrelated to the evolution of the venom apparatus. it is difficult to consider these as aeparau phrnomena. The present report treats of certain chemical and pharmacological properties of snake vrnoma and the relationships between these activities and the development of the vrnom apparatus . BArrrrER, W ., RUSSELL, F . E ., BAATOTI, B . and SctawACeR, E. (Departmrnt of Pharmacology and Toxicology, University of Arizona, Tucson, AZ 85721, U .S .A.) Fatal rattlesnake bic in a dtild. Yet . Hum . Taut. 26, 5 (1984) . A 4-vEAR old had acute onset of vomiting, coma, upper airway obstruction and angioedena while at a roadside area in southeaatem Arizona . He responded to ephinephrine, diphenhydramine and corticosteroids 1 h later ; a diagnosis of anaphylaxis was entertained . Eightern hours later a puncture was noted oa the left calf. He showed rhabdomyolysis with a creatinine kinase of 284,900 IU/l, was responsive to painful stimuli, but had no papillary, rnrneal or doll's eyes reflexes . Wyeth antivenin (13 vials) was administered with reversal of the cranial nerve findings . However, he later became comatose and lost all reflex activity . Five vials of antivenin produced no change . Serial EEG's revealed electrical silrnce and life support efforts were stopped . Post-mortem revealed a single fang mark over the sapahenous vein . There was minimal local reaction, but cardiac and skeletal mends showed evidrnce of rhabdomyolysia . ELISA to multiple specific antibodies was positive in brain tissue to Crotales seutelates vrnom . This cane demonstrates the course of infra- or perivrnous rnvrnomation with nervous system efforts and use of ELISA in identifying the offending culprit . This case was the fast snake venom poisoning case in the United States in which the diagnosis was established by ELISA . The nervous system effects, indudiag the findings at post-mortem, will be described in a neurological journal by Ih Barton . LAt., R . (Ed .) lrtsrcticide Microbiology. Berlin : Springer Verlag (1984) . THE WIDESPREAD and sometimes indiscriminate use of insecticides causes dramatic efforts in the environmrnt affecting non-target organisms to large extrnt . The presrnt book contains trn papers on miaobial-inaectidde