A comparative study of the biological activities of rattlesnake (genera Crotalus and Sistrurus) venoms.

1. The hemorrhagic, procoagulant, anticoagulant, protease, arginine ester hydrolase, phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, hyaluronidase, phospholipase A and L-amino acid oxidase activities of 50 venom samples from 20 taxa of rattlesnake (genera Crotalus and Sistrurus) were examined. 2. The results show that notwithstanding individual variations in the biological activities of Crotalus venoms and the wide ranges of certain biological activities observed, there are some common characteristics at the genus and species levels. 3. The differences in biological activities of the venoms compared can be used for differentiation of the species. Particularly useful for this purpose are the thrombin-like enzyme, protease, arginine ester hydrolase, hemorrhagic and phospholipase A activities and kaolin-cephalin clotting time measurements.     

Inhibition of enzymatic and pharmacological activities of some snake venoms and toxins by Mandevilla velutina (Apocynaceae) aqueous extract

Phospholipases A(2) (PLA(2)) are multifunctional proteins which exhibit varied biological activities correlated to the structural diversities of the sub-classes. The crude aqueous extract from subterranean system of Mandevilla velutina, a plant found in Brazilian savanna, was assayed for its ability to inhibit biological activities of several snake venoms and isolated PLA(2)s. The extract induced total inhibition of the phospholipase activity of Crotalus durissus terrificus venom and only partial inhibition of Bothrops venoms. When assayed against purified toxins, the highest efficacy was detected against CB and crotoxin, while almost ineffective against PLA(2)s from the genus Bothrops. Although M. velutina crude extract significantly inhibited the myotoxic activity of C. d. terrificus venom and CB, it produced only partial inhibition of either Bothrops jararacussu venom or its main myotoxins BthTX-I (basic Lys49), BthTX-II (basic Asp49) and BthA-I-PLA(2) (acidic Asp49). The extract exhibited also full inhibition of hemorrhage caused by Bothrops alternatus, Bothrops moojeni and Bothrops pirajai snake venoms, but partial inhibition (90%) of that induced by B. jararacussu venom. The extract was ineffective to inhibit the fibrinogenolytic activity of B. moojeni, B. alternatus and B. pirajai crude venoms, while their caseinolytic activity was only partially inhibited. No inhibition of the anticoagulant activity, although partial reduction of the edema-inducing activity of C. d. terrificus and B. alternatus crude venoms, CB, PrTX-I, BthTX-I and crotoxin was observed. Besides extending survival of mice injected with lethal doses of C. d. terrificus and B. jararacussu venoms, M. velutina extract decreased to 50% the lethality of mice. Extracts of 18 month old micropropagated plants were able to partially neutralize the effect of the crude venoms and toxins.     

A comparative study of the biological properties of some sea snake venoms.

1. The protease, phosphodiesterase, alkaline phosphomonoesterase, L-amino acid oxidase, acetylcholinesterase, phospholipase A, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, procoagulant, anticoagulant and hemorrhagic activities of ten samples of venoms from seven taxa of sea snakes were examined. 2. The results show that venoms of sea snakes of both subfamilies of Hydrophiinae and Laticaudinae are characterized by a very low level of enzymatic activities, except phospholipase A activity and, for some species, hyaluronidase activity. 3. Because of the low levels of enzymatic activities and the total lack of procoagulant and hemorrhagic activities, venom biological properties are not useful for the differentiation of species of sea snakes. Nevertheless, the unusually low levels of enzymatic activities of sea snake venoms may be used to distinguish sea snake venoms from other elapid or viperid venoms.     

A comparative study of the biological properties of Australian elapid venoms

1. The hemorrhagic, procoagulant, anticoagulant, protease, phosphodiesterase, alkaline phosphomonoesterase, L-amino acid oxidase, acetylcholinesterase, arginine ester hydrolase, phospholipase A, 5'-nucleotidase and hyaluronidase activities of 39 samples of venoms from 13 species (15 taxa) of Australian elapids were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. The results indicate that Australian elapid venoms can be divided into two groups: procoagulant Australian venoms (including N. scutatus, N. ater, O. scutellatus, O. microlepidotus, P. porphyriacus, T. carinatus, H. stephensii and P. textilis) and non-procoagulant Australian venoms (including A. superbus, P. colletti, P. australis, P. guttatus and A. antarcticus). 3. The non-procoagulant Australian venoms exhibited biological properties similar to other elapid venoms, while the procoagulant Australian venoms exhibited some properties characteristic of viperid venoms. 4. The data show that information on venom biological properties can be used for differentiation of many species of Australian elapids. 5. Particularly useful for this purpose are the hyaluronidase, alkaline phosphomonoesterase, acetylcholinesterase, and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.     

Characterization of the snake venoms from seven Brazilian species of Bothrops by FPLC anion-exchange chromatography.

1. The elution profiles and the caseinolytic, myotoxic, coagulant and hemorrhagic activities of the venoms of seven Bothrops species fractionated on a Mono-Q FPLC column were analyzed. 2. Each venom separated into 16-20 peaks, with good reproducibility and the activities were concentrated in virtually discrete regions of the chromatogram. 3. There is a considerable overlap of active proteins in the different species venoms and our results indicate that a venom pool with the species B. jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. atrox venoms would contain the major active proteins determined in the seven species.     

A comparative study of the enzymatic and toxic properties of venoms of the Asian lance-headed pit viper (Genus Trimeresurus)

1. The lethalities, anticoagulant effects, hermorrhagic, thrombin-like enzyme, hyaluronidase, protease, arginine ester hydrolase, 5'-nucleotidase, L-amino acid oxidase, alkaline phosphomonoesterase, phosphodiesterase and phospholipase A activities of twenty-three samples of venoms from twelve species of Asian lance-headed pit vipers (genus Trimeresurus) were examined. 2. The results indicate that notwithstanding individual variations in venom properties, the differences in biological properties of the Trimeresurus venoms can be used for the differentiation of venoms from different species of Trimeresurus. 3. The results also suggest that differences in the biological properties of snake venoms are useful parameters in the classification of snake species. 4. Our results indicate that venoms from the species T. okinavensis exhibited biological properties markedly different from other Trimeresurus venoms examined. This observation supports the recently proposed reclassification of T. okinavensis as a member of the genus Ovophis, rather than the genus Trimeresurus.     

Prey specificity, comparative lethality and compositional differences of coral snake venoms

Toxicities of crude venoms from 49 coral snake (Micrurus sp.) populations, representing 15 nominal taxa, were examined in both laboratory mice and in native prey animals and compared with data gathered from two non-micrurine elapids and a crotalid, which served as outgroups. These venoms were further compared on the basis of 23 enzymatic activities. Both toxicities and enzymatic activities were analyzed with respect to natural prey preferences, as determined from stomach content analyses and literature reports. Venoms of nearly all Micrurus for which prey preferences are known, are more toxic to natural prey than to non-prey species. Except for amphisbaenians, prey are more susceptible to venoms of Micrurus that feed upon them, than to venoms of those that eat other organisms. All venoms were more toxic i.v.>i.p.>i.m. Route-specific differences in toxicity are generally greatest for preferred prey species. Cluster analyses of venom enzymatic activities resulted in five clusters, with the fish-eating M. surinamensis more distant from other Micrurus than even the crotalid, Bothrops moojeni. Ophiophagous and amphisbaenian-eating Micrurus formed two close subclusters, one allied to the outgroup species Naja naja and the other to the fossorial, ophiophagous Bungarus multicinctus. Prey preference is shown to be the most important determinant of venom composition in Micrurus.     

Comparative enzymatic composition of Brazilian coral snake (Micrurus) venoms.

1. Venoms of 11 coral snake taxa, including Micrurus albicinctus, M. corallinus, M. frontalis altirostris, M. f. brasiliensis, M. f. frontalis, M. fulvius fulvius, M. ibiboboca, M. lemniscatus ssp., M. randonianus, M. spixii spixii, and M. surinamensis surinamensis, were examined for 13 enzymatic activities. 2. These were compared with venoms of three outgroup taxa: Naja naja kaouthia, Bungarus multicinctus, and Bothrops moojeni. 3. Enzyme activity levels in Micrurus venoms were highly variable from species to species. 4. All venoms possessed phospholipase activity. 5. Protease activity against synthetic or dyed natural substrates was generally negligible in all elapid venoms examined. By contrast, most Micrurus venoms displayed ample L-leucine aminopeptidase activity. 6. Venom of M.s. surinamensis was significantly different from those of its congeners in most assays.     

A comparative study of the biological properties of krait (genus Bungarus) venoms

1. The intravenous median lethal doses (LD50), protease, phosphodiesterase, alkaline phosphomonoesterase, L-amino acid oxidase, acetylcholinesterase, phospholipase A, 5'-nucleotidase, hyauronidase and anticoagulant activities of fourteen samples of venoms from the four common species of krait (Bungarus caeruleus, Bungarus candidus, Bungarus multicinctus and Bungarus fasciatus) were examined. 2. The results indicate that even though there are individual variations in the biological properties of the krait venoms, interspecific differences in the properties can be used for differentiation of the venoms from the four species of Bungarus. Particularly useful for this purpose are the LD50's and the contents of 5'-nucleotidase and hyaluronidase of the venoms.     

Neuromuscular action of venom from the South American colubrid snake Philodryas patagoniensis

Snakes of the opisthoglyphous genus Philodryas are widespread in South America and cause most bites by colubrids in this region. In this study, we examined the neurotoxic and myotoxic effects of venom from Philodryas patagoniensis in biventer cervicis and phrenic nerve-diaphragm preparations and we compared the biochemical activities of venoms from P. patagoniensis and Philodryas olfersii. Philodryas patagoniensis venom (40 microg/mL) had no effect on mouse phrenic nerve-diaphragm preparations but caused time-dependent neuromuscular blockade of chick biventer cervicis preparations. This blockade was not reversed by washing. The highest concentration of venom tested (40 microg/mL) significantly reduced (p<0.05) the contractures to exogenous acetylcholine (55 microM and 110 microM) and K(+) (13.4 mM) after 120 min; lower concentrations of venom had no consistent or significant effect on these responses. Venom caused a concentration- and time-dependent release of creatine kinase (CK) from biventer cervicis preparations. Histological analysis showed contracted muscle fibers at low venom concentrations and myonecrosis at high concentrations. Philodryas venoms had low esterase and phospholipase A(2) but high proteolytic activities compared to the pitviper Bothrops jararaca. SDS-PAGE showed that the Philodryas venoms had similar electrophoretic profiles, with most proteins having a molecular mass of 25-80 kDa. Both of the Philodryas venoms cross-reacted with bothropic antivenom in ELISA, indicating the presence of proteins immunologically related to Bothrops venoms. RP-HPLC of P. patagoniensis venom yielded four major peaks, each of which contained several proteins, as shown by SDS-PAGE. These results indicate that P. patagoniensis venom has neurotoxic and myotoxic components that may contribute to the effects of envenoming by this species.     

A comparative study of the biological properties of venoms from snakes of the genus Vipera (true adders)

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 26 samples of venoms of 13 taxa of Vipera were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. The results indicate the presence of certain common characteristics among the venoms, particularly if V. russelli is excluded from the comparison. The results also support the recently proposed reassignment of V. russelli to a separate genus. 3. The data show that information on venom biological properties can be used for differentiation of venoms of many species of Vipera. Particularly useful for this purpose are the protease, phosphodiesterase, phospholipase A and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.     

Snake venomics of the Lesser Antillean pit vipers Bothrops caribbaeus and Bothrops lanceolatus: correlation with toxicological activities and immunoreactivity of a heterologous antivenom

The venom proteomes of the snakes Bothrops caribbaeus and Bothrops lanceolatus, endemic to the Lesser Antillean islands of Saint Lucia and Martinique, respectively, were characterized by reverse-phase HPLC fractionation, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venoms contain proteins belonging to seven ( B. caribbaeus) and five ( B. lanceolatus) types of toxins. B. caribbaeus and B. lanceolatus venoms contain phospholipases A 2, serine proteinases, l-amino acid oxidases and zinc-dependent metalloproteinases, whereas a long disintegrin, DC-fragments and a CRISP molecule were present only in the venom of B. caribbaeus, and a C-type lectin-like molecule was characterized in the venom of B. lanceolatus. Compositional differences between venoms among closely related species from different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. The venoms of these two species differed in the composition and the relative abundance of their component toxins, but they exhibited similar toxicological and enzymatic profiles in mice, characterized by lethal, hemorrhagic, edema-forming, phospholipase A 2 and proteolytic activities. The venoms of B. caribbaeus and B. lanceolatus are devoid of coagulant and defibrinogenating effects and induce only mild local myotoxicity in mice. The characteristic thrombotic effect described in human envenomings by these species was not reproduced in the mouse model. The toxicological profile observed is consistent with the abundance of metalloproteinases, PLA 2s and serine proteinases in the venoms. A polyvalent (Crotalinae) antivenom produced in Costa Rica was able to immunodeplete approximately 80% of the proteins from both B. caribbaeus and B. lanceolatus venoms, and was effective in neutralizing the lethal, hemorrhagic, phospholipase A 2 and proteolytic activities of these venoms.     

A comparative study of the biological properties of venoms of some old world vipers (subfamily viperinae).

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, hyaluronidase, alkaline phosphomonoesterase, 5'-nucleotidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 30 samples of venoms from nine species (12 taxa) of the old world vipers (Subfamily Viperinae) including snakes from the genera Bitis, Causus, Cerastes, Echis, Eristicophis and Pseudocerastes, were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. Examination of the biological properties of the venoms of the Viperinae tested indicates the presence of common venom biological characteristics at the various phylogenic levels. 3. Venoms of most species of the Viperinae examined exhibited characteristic biological properties at the species level, and this allows the differentiation of the Viperinae species by differences in their biological properties. 4. Particularly useful for this purpose, are the effects of venom on kaolin-cephalin clotting time of platelet poor rabbit plasma and the Sephadex G-75 gel filtration pattern and arginine ester hydrolase activity of the venom.     

Biological and immunological properties of the venom of Bothrops alcatraz, an endemic species of pitviper from Brazil

Bothrops alcatraz is a new pitviper species derived from the Bothrops jararaca group, whose natural habitat is situated in Alcatrazes Archipelago, a group of marine islands near São Paulo State coast in Brazil. Herein, the biological and biochemical properties of venoms of four adult specimens of B. alcatraz were examined comparatively to a reference pool of Bothrops jararaca venom. Both venoms showed similar activities and electrophoretic patterns, but B. alcatraz venom showed three protein bands of molecular masses of 97, 80 and 38 kDa that were not present in B. jararaca reference venom. The i.p. median lethal dose of B. alcatraz venom ranged from 5.1 to 6.6 mg/kg, while it was 1.5 mg/kg for B. jararaca venom. The minimum hemorrhagic dose of B. jararaca venom was 0.63, whereas 2.28 μg/mouse for B. alcatraz venom. In contrast, B. alcatraz venom was more potent in regard to procoagulant and proteolytic activities. These differences were supported by western blotting and neutralization tests, employing commercial bothropic antivenom, which showed that hemorrhagic and lethal activities of B. alcatraz venom were less effectively inhibited than B. jararaca venom. Such results evidence that B. alcatraz shows quantitative and qualitative differences in venom composition in comparison with its B. jararaca relatives, which might represent an optimization of venom towards a specialized diet.     

Angiostatin-like molecules are generated by snake venom metalloproteinases

Angiostatin is a plasminogen-derived anti-angiogenic factor composed of its first four kringle structures. This molecule is generated by proteolytic cleavage of plasminogen by some proteolytic enzymes in vitro. Since venoms of viper snakes are a rich source of both serine- and metalloproteinase, we hypothesized that angiostatin-like polypeptides could be generated during the envenomation after snake bites and play a pathophysiological role in the local tissue damage and regeneration. Our results showed that crude venoms from several species of Bothrops snakes were able to generate angiostatin-like polypeptides and purified metalloproteinases but not serine proteinases from Bothrops jararaca and Bothrops moojeni venoms were responsible for their generation in vitro. The putative plasminogen cleavage sites by the crude venoms and purified proteinases were determined by N-terminal amino acid sequencing of the angiostatin-like molecules. Angiostatin-like peptides derived from human plasminogen digestion by jararhagin, a metalloproteinase isolated from B. jararaca venom, inhibited endothelial cell proliferation in vitro. These results indicate that angiostatin-like molecules can be generated upon snakebite envenomations and may account for the poor and incomplete regenerative response observed in the damaged tissue.     

Profilings of subproteomes of lectin-binding proteins of nine Bothrops venoms reveal variability driven by different glycan types

Snake venom proteomes have long been investigated to explore a multitude of biologically active components that are used for prey capture and defense, and are involved in the pathological effects observed upon mammalian envenomation. Glycosylation is a major protein post-translational modification in venoms and contributes to the diversification of proteomes. We have shown that Bothrops venoms are markedly defined by their content of glycoproteins, and that most N-glycan structures of eight Bothrops venoms contain sialic acid, while bisected N-acetylglucosamine was identified in Bothrops cotiara venom. To further investigate the mechanisms involved in the generation of different venoms by related snakes, here the glycoproteomes of nine Bothrops venoms (Bothrops atrox, B. cotiara, Bothrops erythromelas, Bothrops fonsecai, B. insularis, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi) were comparatively analyzed by enrichment with three lectins of different specificities, recognizing bisecting N-acetylglucosamine- and sialic acid-containing glycoproteins, and mass spectrometry. The lectin capture strategy generated venom fractions enriched with several glycoproteins, including metalloprotease, serine protease, and L- amino acid oxidase, in addition to various types of low abundant enzymes. The different contents of lectin-enriched proteins underscore novel aspects of the variability of the glycoprotein subproteomes of Bothrops venoms and point to the role of distinct types of glycan chains in generating different venoms by closely related snake species.     

Nucleotidase and DNase activities in Brazilian snake venoms

Among the myriad of enzymes present in animal venoms, nucleotidases and nucleases are poorly investigated. Herein, we studied such enzymes in 28 crude venoms of animals found in Brazil. Higher levels of ATPase, 5'-nucleotidase, ADPase, phosphodiesterase and DNase activities were observed in snake venoms belonging to Bothrops, Crotalus and Lachesis genera than to Micrurus genus. The venom of Bothrops brazili snake showed the highest nucleotidase and DNase activities, whereas that of Micrurus frontalis snake the highest alkaline phosphatase activity. On the other hand, the venoms of the snake Philodryas olfersii and the spider Loxosceles gaucho were devoid of most nucleotidase and DNase activities. Species that exhibited similar nucleotidase activities by colorimetric assays showed different banding pattern by zymography, suggesting the occurrence of structural differences among them. Hydrolysis of nucleotides showed that 1 mol of ATP is cleaved in 1 mol of pyrophosphate and 1 mol of orthophosphate, whereas 1 mol of ADP is cleaved exclusively in 2 mol of orthophosphates. Pyrophosphate is barely hydrolyzed by snake venoms. Phosphodiesterase activity was better correlated with 5'-nucleotidase, ADPase and ATPase activities than with DNase activity, evidencing that phosphodiesterases are not the main agent of DNA hydrolysis in animal venoms. The omnipresence of nucleotidase and DNase activities in viperid venoms implies a role for them within the repertoire of enzymes involved in immobilization and death of preys.     

A comparative study of the biological activities of venoms from snakes of the genus Agkistrodon (moccasins and copperheads)

1. The hemorrhagic, procoagulant, anticoagulant, phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, hyaluronidase, arginine ester hydrolase, phospholipase A, L-amino acid oxidase and protease activities of 31 samples of venom from three species of Agkistrodon (A. bilineatus, A. contortrix and A. piscivorus) and 10 venom samples from five other related species belonging to the same tribe of Agkistrodontini were examined. 2. The results indicate that interspecific differences in certain biological activities of the Agkistrodon venoms are more marked than individual variations of the activities, and that these differences can be used for differentiation of the species. Particularly useful for this purpose are the phosphodiesterase, arginine ester hydrolase and anticoagulant activities of the venoms. 3. Venoms of the subspecies of A. contortrix and A. piscivorus do not differ significantly in their biological activities.     

Biological and immunological characteristics of the poison of Bothrops cotiara (Serpentes: Viperidae)

Bothrops cotiara is a venomous snake sporadically found in the province of Misiones in Argentina, South of Brazil and Paraguay. Data on the clinics of the envenomation produced by its bite and on its venom are scarce. There is no information on the neutralizing capacity of the antivenoms available. In this study, the lethal potency, hemorrhagic, necrotizing, coagulant and thrombin-like, defibrinogenating, indirect hemolytic and fibrinolytic activities of the venom of B. cotiara specimens from the province of Misiones were determined. The toxic activities were within the range of those described for the other Bothrops species from Argentina, and the electrophoretic and chromatographic studies showed similarities with those described for the other bothropic venoms. The immunochemical reactivity of six South American anti Viper antivenoms (ELISA) have a strong reactivity with all the antivenoms studied. The neutralizing capacity of three of these therapeutic antivenoms against the lethal potency and hemorrhagic, necrotizing, coagulant, thrombin-like and hemolytic activities showed a very close neutralizing capacity. Our data strongly suggest that the antivenoms for therapeutic use available in this area of South America are useful to neutralize the toxic and enzymatic activities of the venom of this uncommon specie of Bothrops.     

A comparative study of the biological properties of Dendroaspis (mamba) snake venoms.

1. The biological properties of twelve samples of venoms from all four species of Dendroaspis (mamba) were investigated. 2. Dendroaspis venoms generally exhibited very low levels of protease, phosphodiesterase and alkaline phosphomonoesterase; low to moderately low level of 5'-nucleotidase and very high hyaluronidase activities, but were devoid of L-amino acid oxidase, phospholipase A, acetylcholinesterase and arginine ester hydrolase activities. The unusual feature in venom enzyme content can be used to distinguish Dendroaspis venoms from other snake venoms. 3. All Dendroaspis venoms did not exhibit hemorrhagic or procoagulant activity. Some Dendroaspis venoms, however, exhibited strong anticoagulant activity. The intravenous median lethal dose of the venoms ranged from 0.5 microgram/g mouse to 4.2 micrograms/g mouse. 4. Venom biological activities are not very useful for the differentiation of the Dendroaspis species. The four Dendroaspis venoms, however, can be differentiated by their venom SDS-polyacrylamide gel electrophoretic patterns.