Neutralization of kinin-releasing enzymes of crotalid venoms by monospecific and polyspecific antivenoms

The amounts of kinin-releasing enzymes in the venoms of Crotalus atrox, Crotalus adamanteus, Crotalus scutulatus scutulatus and Agkistrodon piscivorus piscivorus were measured by determining the amounts of kinin released from a sheep kininogen substrate by means of a specific radioimmunoassay. Four monospecific and two commercial polyspecific antivenom IgG samples were tested for their ability to reduce the kinin-releasing activities of the four crotalid venoms measured in vitro. All of the antivenom IgG samples were able to neutralize venom kininogenase activity to varying extents. On of the commercial polyspecific antivenoms was of equal or higher potency than the corresponding monospecific antivenoms for three of the venoms tested, indicating a high degree of cross-neutralization. F(ab')2 and especially Fab fragments of that polyspecific antivenom IgG were also effective in reducing the kinin-releasing activities of the four crotalid venoms.     

Procoagulant activities in venoms from central Asian snakes.

The venoms from central Asian snakes (Echis carinatus, Echis multisquamatus, Vipera ursini, Vipera lebetina, Agkistrodon halys halys and Naja naja oxiana) contain several enzymes with amidolytic- and procoagulant activity. We have characterized the activities and the mol. wts of the venom enzymes that are able to convert a number of commercially available chromogenic substrates for activated coagulation factors. The chromogenic substrate cleavage patterns obtained for the crude venoms may be helpful tools in the further identification of venom fractions and venom enzymes with procoagulant activity. The crude venoms were also tested for their ability to clot fibrinogen, to lyse fibrin polymers and to activate the coagulation factors prothrombin, factor X and factor V. The products of venom-catalyzed coagulation factor activation were structurally characterized by SDS gel electrophoresis and were compared with activated coagulation factors that are generated under physiological conditions.     

Critical comments on the quality of some commercial antivenins

Five commercial antivenins were examined for their ability to neutralise the venoms obtained from species of continental European snakes. Purity of the serum preparations was examined by paper electrophoresis. Immunoelectrophoresis and immunodiffusion methods were used to determine the neutralising capacity of the sera against the venom of Vipera aspis, Vipera berus, Vipera ammodytes and Bitis lachesis. Only one serum gave a reaction with all venoms and was thus shown to be highly concentrated and purified.     

Studies on hemorrhagic toxins from the venoms of Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis and Agkistrodon acutus and arginine ester hydrolases from Trimeresurus mucrosquamatus venom

Venom samples were corrected from several poisonous snakes, such as Bungarus multicinctus, Trimeresurus mucrosquamatus, T. gramineus, T. flavoviridis, and Agkistrodon acutus, and stored in a desiccator at room temperature for 25 to 31 years. Then they were compared with fresh venoms as to their biological activities. The characteristic local symptoms produced by the bite of venomous snakes of Crotalidae and Viperidae are hemorrhage, necrosis and muscular degeneration. Hemorrhagic toxins were purified from Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis, and Agkistrodon acutus venoms and their biological, biochemical, and pathological properties were investigated. Arginine ester hydrolases are present in the venoms of Crotalidae and Viperidae, but are not found in the venoms of Elapidae and Hydrophiidae. In this paper we describe the enzymatic and biological activities of arginine ester hydrolases from a Trimeresurus mucrosquamatus venom.     

Variation in yield and lethality of venoms from Iranian snakes

The dangerous venomous terrestrial snakes of Iran belong to three groups: the Elapidae (cobras); the Viperinae (true vipers); the Crotalinae (pit vipers). Geographical distribution of each species was determined. Studies on the venoms extracted from the following Iranian snakes, Oxus cobra, Naja naja oxiana, Levantine viper (Afyi), Vipera lebetina, Carpet viper, Echis carinatus, Persian horned viper, Pseudocerastes persicus, Latifii viper, Vipera latifii, Mountain viper, Vipera xanthina and Caucasus pit viper (Agkistrodon halys), indicated that the yield of venom varies in each species. Venoms were compared for their lethality (i.v. LD50 in mice) and their rate of production. The antigenic components of the venoms were compared with their antisera by gel diffusion tests. To obtain the best results from antivenom treatment, the serum should be made against the venom of the local population of snakes or, at least, the commercial antivenom should be controlled for potency by testing with local reference venom.     

Physiological and biochemical properties of A kallikrein-like enzyme from the venom of Vipera aspis aspis (aspic viper)

and . Physiological and biochemical properties of a kallikrein-like enzyme from the venom of Vipera aspis aspis (aspic viper), Toxicon 26, 1193–1204, 1988.—A kallikrein-like enzyme was isolated from the venom of Vipera aspis aspis by Sephadex G-75, Q-Sepharose and Heparin-Sepharose CL-6B column chromatography. The purified enzyme is a glycoprotein with a mol.wt of 43,000 and an isoelectric point of 4.1. The enzyme possesses arginine ester hydrolase activity, but no proteolytic activity against either dimethylcasein or fibrinogen. The reaction mixture of the enzyme and bovine plasma induced contraction of the isolated rat uterus, suggesting that the enzyme releases kinin from the plasma constituent. The amount of enzyme, which releases an equal amount of kinin corresponding to 1 nmole of bradykinin per min, is 2.36 mg. Additionally, the kallikrein-like enzyme demonstrated capillary permeability-increasing activity and hypotensive activity. A synthetic kininogen analog, Ser-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser, was cleaved by the enzyme to release bradykinin and kallidin, also indicating that the enzyme has a kallikrein-like activity. Uterine contraction, capillary permeability-increasing activity and arginine ester hydrolase activity were inhibited by diisopropyl fluorophosphate, suggesting that the serine hydroxyl group is essential for enzymatic and biological activities. Antithrombin III and heparin, serine-protease inhibitors found in plasma had no inhibitory effect on these activities of the purified enzyme. The amino acid sequence of the NH₂ terminal region of the enzyme has similarities with kallikrein-like enzynes from other snake venoms and with porcine pancreatic kallikrein.     

Proteolytic activity of tarantula venoms due to contamination with saliva

The venoms of the tarantulas Pterinochilus sp., Aphonopelma chalcodes and Dugesiella hentzi, collected by electroshock, were tested for proteolytic activity. The proteases found were chymotrypsin-like with optima of Hide Powder Azure hydrolysis at approximately pH 8. However, activity inconsistencies led to the discovery that the presence of proteolytic enzymes in the three venoms was the result of a contamination with fluids utilized for external digestion by the spiders. Contamination of the venoms could be prevented by forcing the spider to strike through a membrane prior to stimulation. Based on the findings of a literature survey, it is concluded that previous demonstrations of proteolytic activities of spider venoms might have resulted from contamination by saliva.     

Chemistry and Biochemistry of Kallikrein-Like Enzyn from Snake Venoms

Abstract

The chemical and physiological properties of various kallikrein-like (kinin-releasing) enzymes from Viperidae and Crotalidae snake venoms have been compared. These enzymes act on plasma kininogen to release the potent vasodepressors bradykinin and/or kallidin. Moreover, they hydrolyze the vasopressor angiotensin II and its precursor angiotensin I. Although fibrinogen clotting ability is absent from kallikrein-like enzymes, most of these enzymes degrade the peptide chains of fibrinogen. Considerable sequence homology occurs between kallikrein-like enzymes and thrombin-like enzymes, however physiological substrate specificities of the two enzymes differ. Similarities and differences in the structure and function of these enzymes is discussed.     

Comparative study of the venoms of three subspecies of Lachesis muta (bushmaster) from Brazil, Colombia and Costa Rica

A comparative study was performed on the pharmacology and biochemistry of venoms from three subspecies of Lachesis muta (L. m. stenophrys, L. m. muta and L. m. rhombeata) from Brazil, Colombia and Costa Rica. All venoms induced lethal, hemorrhagic, edema-forming, myotoxic, coagulant and defibrinating effects, showing also proteolytic and indirect hemolytic activities. The venoms of L. m. stenophrys from Costa Rica and L. m. muta from Cascalheira, Brazil, had the highest lethal and hemorrhagic activities and the venom of L. m. rhombeata showed the highest coagulant activity, whereas no significant differences were observed in myotoxic and edema-forming activities at most of the time intervals studied. In addition, venoms showed similar electrophoretic patterns on SDS–polyacrylamide gel electrophoresis. In conclusion, despite quantitative differences in toxic and enzymatic activities, together with subtle variations in electrophoretic patterns, our results indicate that experimental envenomation by these venoms induce a qualitatively similar pathophysiological profile.     

Collagenolytic activity of snake and spider venoms

The venoms of the snakes B. atrox, B. Jararaca, C. durissus, A. piscivorus, and the spiders P. fera, L. erythrognatha possess proteolytic activity which attacks gelatine, casein and azocoll. Denatured (heat, urea) collagen is digested too. However, native collagen is not attacked by the enzymes present in the venoms investigated. Following subcutaneous injection of A. piscivorus venom, a statistically significant increase in urinary hydroxyproline excretion occurred. This finding indicates a degradation of collagen by animal venoms in vivo. The mechanism of this action remains unknown.     

Cross-neutralization of thrombin-like enzymes in snake venoms by polyvalent antivenoms

and . Cross-neutralization of thrombin-like enzymes in snake venoms by polyvalent antivenoms. Toxicon 27, 1397–1399, 1989.—Five polyvalent antivenoms (Crotalidae; Orient, North, Central and South Africa) were tested for their ability to neutralize the thrombin-like activity of snake venoms (Bitis gabonica, Agkistrodon acutus, Bothrops asper, B. atrox, Crotalus adamanteus). Considerable cross-neutralization was observed. Anti-coagulase antibodies were isolated from an antivenom by affinity chromatography using a purified enzyme from Bitis gabonica venom. These antibodies neutralized the activity of most snake venom coagulant enzymes.     

Characterization of venom (Duvernoy’s secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins

R.E. Hill and S.P. Mackessy. Characterization of venom (Duvernoy’s secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins. Toxicon XX, xx–yy, 2000. — Venomous colubrids, which include more than 700 snake species worldwide, represent a vast potential source of novel biological compounds. The present study characterized venom (Duvernoy’s gland secretion) collected from twelve species of opisthoglyphous (rear-fanged) colubrid snakes, an extremely diverse assemblage of non-venomous to highly venomous snakes. Most venoms displayed proteolytic activity (casein), though activity levels varied considerably. Low phosphodiesterase activity was detected in several venoms (Amphiesma stolata, Diadophis punctatus, Heterodon nasicus kennerlyi, H. n. nasicus and Thamnophis elegans vagrans), and acetylcholinesterase was found in Boiga irregularis saliva and venom, but no venoms displayed hyaluronidase, thrombin-like or kallikrein-like activities. High phospholipase A₂ (PLA₂) activity was found in Trimorphodon biscutatus lambda venom, and moderate levels were detected in Boiga dendrophila and D. p. regalis venoms as well as B. dendrophila and H. n. nasicus salivas. Non-reducing SDS–PAGE revealed 7–20 protein bands (3.5 to over 200 kD, depending on species) for all venoms analyzed, and electrophoretic profiles of venoms were typically quite distinct from saliva profiles. Components from A. stolata, Hydrodynastes gigas, Tantilla nigriceps and T. e. vagrans venoms showed protease activity when run on gelatin zymogram gels. N-terminal protein sequences for three 26 kD venom components of three species (H. gigas, H. torquata, T. biscutatus) and one 3.5 kD component (T. nigriceps) were also obtained, and the 3.5 kD peptide showed apparent sequence homology with human vascular endothelial growth factor; these data represent the first sequences of colubrid venom components. Protease, phosphodiesterase and PLA₂ activities are also common to elapid and viperid snake venoms, but it is apparent that numerous other (as yet undescribed) components make up the majority of colubrid venom proteins. The complex nature of venoms produced by most species surveyed, and the high levels of protease or phospholipase A₂ activity of some venoms, suggest that many colubrids could become an important source of human health concern as encounters with these snakes increase.     

Heterogeneity of Echis venoms from different sources

R. C. . Heterogeneity of Echis venoms from different sources. Toxicon 25, 1343 – 1346, 1987. — The heterogeneity of separate Echis carinatus and Echis coloratus venom lots were evaluated by high performance size-exclusion chromatography, as well as by clotting and esterolytic activities. Each lot of E. carinatus and E. coloratus venoms appeared to be unique. The variation in E. carinatus venom activities from snakes within the same species could lead to contradictory biochemical and/or physiological test results.     

Neutralization of different activities of venoms from nine species of Bothrops snakes by Bothrops jararaca antivenom.

Antivenoms are the usual treatment in cases of systemic envenoming by Bothrops snakes. However, the neutralization of each venom component by the antivenom is not well established. Bothrops jararaca antivenom, produced in rabbits, recognizes the venoms of nine different Bothrops species with high ELISA antibody titres. Western blot analysis showed that almost all antigens present on both homologous and heterologous venoms are recognized. Neutralization tests were performed using whole antivenom or its IgG fraction. The antivenom was able to neutralize the haemorrhagic, coagulant and necrotizing activities of the heterologous venoms in the same antivenom/venom proportion as for the homologous venom. Myotoxic activity was only partially neutralized. Neutralization of the proteolytic activity of heterologous venoms required higher amounts of antivenom than for the homologous venom. Phospholipase and oedema-inducing activities were completely neutralized only in the homologous system.     

Immunological comparison of phospholipases A2 present in rattlesnake (genus Crotalus) venoms

The immunological properties of phospholipase A₂ present in venoms from seven species of rattlesnakes (genus Crotalus) from different geographical distributions have been compared. Rabbit antiserum to purified PLA₂ of Crotalus scutulatus salvini venom was used, employing inhibition of enzymic activity and immunodiffusion techniques. The amount of antiserum required for 50% inhibition of the PLA₂ activity in the venoms varied from slightly more than that required for inhibition of the activity in the homologous venom to a large amount (effectively zero inhibition). A particularly close relationship in the antigenic makeup was observed between the phospholipases of C. s. salvini and C. atrox venoms as evidenced by inhibition of enzymic activity and immunodiffusion studies. No immunological cross reactivity was observed between the enzymes of the subspecies C. s. salvini and C. s. scutulatus. The results suggest extensive variations in the antigenic structure of the phospholipase molecules. The findings may also have some taxonomic significance.     

Characterisation of the biochemical and biological variations from the venom of the death adder species (Acanthophis antarcticus, A. praelongus and A. pyrrhus)

We report on species variation in the venoms of the three species of death adder; the Common death adder (Acanthophis antarcticus), the Northern death adder (Acanthophis praelongus) and the Desert death adder (Acanthophis pyrrhus). The venoms were found to vary in their biochemical (chromatography) and biological (PLA₂ activity, anticoagulant activity and reactivity with commercial death adder antivenom) properties. Each species produced significant differences in the profile and distribution of PLA₂ activity, when whole venom was applied to a cation-exchange Mono-S column. PLA₂ enzymes were purified from each venom and termed acanthoxin B (from A. praelongus), acanthoxin C (from A. pyrrhus) and the previously characterised acanthoxin A (from A. antarcticus). Acanthoxin B and C showed lower enzymatic activities than acanthoxin A (4.0, 13.7 and 23.9 μmol of phospholipid hydrolyzed/min/mg protein, respectively). N-terminal sequencing revealed acanthoxin B to share highest homology with the numerous PLA₂ isozymes (Pa-12C, Pa-1G, Pa-12A) from the King brown snake (Pseudechis australis) and Acanthin I from the Common death adder. Similar to acanthoxin A, acanthoxin C showed highest homology with Acanthin I/II, and pseudexin A-chain from the Red-bellied black snake (Pseudechis porphyriacus). Whole venom from A. antarcticus, A. praelongus and A. pyrrhus each showed weak anticoagulant activity (being able to prolong coagulation of the plasma for 107, 220 and 195 s, respectively). By immunodiffusion, each venom produced precipitation bands against commercial death adder antivenom.     

Phospholipase A and B activities of reptile and Hymenoptera venoms

Phospholipase B (PLB) activity was found in a variety of snake, Gila monster and Hymenoptera venoms. Hymenoptera venoms, except bee, are an especially rich source of PLB. This was shown by incubation of crude venoms with lysophosphatidylcholine and subsequent titration of liberated fatty acids. PLB activity was not found in pure - or β-bungarotoxin, -cobrotoxin, or one crude snake venom (Crotalus horridus horridus). Snake, Gila monster and bee venoms exhibit higher PLB activity at an alkaline pH, while hornet venom PLB activity is greater at a neutral pH. Phospholipase A₂ (PLA₂) activity was determined using phosphatidylcholine and egg yolk as substrates. In reptile and bee venoms PLA₂ activity is much higher than PLB activity. In contrast, hornet venom PLA₂ and PLB activities are nearly equal. In reptile venoms PLA₂ activity is optimal at neutral pH, in contrast to PLB activity, suggesting that separate proteins or active sites are responsible for PLA₂ and PLB activities. The effect of boiling or heating venoms in either an acidic or alkaline milieu upon subsequent PLA₂ and PLB activity, measured at 37°C, was examined. The PLA₂ and PLB activities of all venoms tested were lost upon boiling at pH 9.4, except for the PLA₂ activity of Notechis scutatus venom which retained about 30% of its activity. Boiling at pH 5.5 results in greatly varying extents of retention of PLA₂ and PLB activities, dependent upon the venom examined. Therefore, the heat stability characteristics for each venom must be experimentally determined, not assumed. Boiling destroys PLA₂ and PLB activities of oriental hornet venom at about the same rate. No conclusive results were obtained from the heating studies as to whether PLA₂ and PLB activities reside upon the same molecule. However, PLA₂ and PLB activities of oriental hornet venom were separated using triple tandem column gel permeation chromatography, demonstrating the existence of two separate proteins for these activities.     

Some enzymic activities of two Australian ant venoms: A jumper ant Myrmecia pilosula and a bulldog ant Myrmecia pyriformis

Venoms from two related Australian ants, a jumper ant (Myrmecia pilosula) and a bulldog ant (Myrmecia pyriformis), were quantitatively analysed for the following enzymic activities: phospholipase A₂, phospholipase B, phospholipase C, hyaluronidase, esterase, acid phosphatase, alkaline phosphatase and phosphodiesterase. Both venoms contained phospholipase A₂, phospholipase B, hyaluronidase, acid phosphatase and alkaline phosphatase activities. Myrmecia pyriformis venom had significantly greater phospholipase B, acid phosphatase and alkaline phosphatase activities than Myrmecia pilosula venom. No detectable quantities of phospholipase C, esterase or phosphodiesterase activities were found in either venom.     

Characterization and cDNA cloning of aminopeptidase A from the venom of Gloydius blomhoffi brevicaudus

The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)₁₈-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile.     

Effect of different Egyptian venoms on the oxygen consumption of isolated tissue slices

The oxygen uptake by slices of albino rat brain (cerebral) hemispheres, cardiac, skeletal and plain (intestine) muscle tissues were studied in Warburg flasks in the presence of 3 mg doses of Naja haje, N. nigricollis, Walterinnesia aegyptia, Cerastes cerastes, and C. vipera venoms added in vitro. Statistically significant inhibition of oxygen uptake by brain tissue resulted in the case of all five venoms. No statistically significant inhibition of oxygen uptake was observed in skeletal muscle tissue slices in the presence of the venoms. Statistically significant inhibition of oxygen uptake was observed in cardiac and in plain muscle tissue slices in the presence of N. nigricollis and W. aegyptia venoms only but not in the presence of the other three venoms.