Proteolytic activities of cobra venoms based on inactivation of alpha 2-macroglobulin

The venoms of various cobra species showed a wide range of abilities to cleave hide powder azure, with Naja naja kaouthia and Ophiophagus hannah venoms showing the lowest activities and Naja nivea venom showing the greatest activity on this dye-linked substrate. The activities of the venoms on hide powder did not completely correlate with their ability to inactivate the alpha 2-macroglobulin of human serum. Incubation of 4-5 micrograms of Naja nigricollis venom per microliter of serum for 30 min caused loss of 95% of the alpha 2-macroglobulin activity of the serum. The inactivation was rapid, reaching 80% inactivation 5 min after mixing. This loss of alpha 2-macroglobulin activity was used to quantitate the weak proteolytic activity of N. nigricollis venom and a partially purified sample of the major fibrinogenolytic proteinase of the venom. The inactivation of alpha 2-macroglobulin was also used to compare the proteinase activities of venoms from seven species or subspecies of cobra. Based on alpha 2-macroglobulin inactivation, N. nigricollis had the highest proteinase activity among the tested venoms. The measurement of alpha 2-macroglobulin inactivation should provide a useful alternative to hide powder digestion for demonstration of weak proteolytic activities in venoms.     

Proteins toxic to arthropods in the venom of elapid snakes.

It has been found that the lethal action of elapid snake venoms to arthropods (fly larvae and isopods) is due to proteic factors differing from the toxins which are strongly and specifically active on mammals.This conclusion was based on the following: (1) Lack of any correlation between the toxic activity on larvae, isopods, and mice of ten elapid snake venoms. (2) Absence of any toxicity to arthropods in pure toxins isolated and purified from several elapid snake venoms according to their lethality. (3) Electrophoretical separation of the venom of the snake Naja mossambica mossambica (= N. nigricollis mossambica) resulted in fractions active either to arthropods and/or to mice. (4) Separation of the above venom by gel filtration on Sephadex G-50 enabled the isolation of fractions highly toxic to arthropods. (5) The above fractions demonstrated a high phospholipase activity corresponding to about 80 per cent of the total activity of the whole venom. The link between phospholipase and toxicity to arthropods will serve as a target for further investigation.It appears that the phenomenon of diversity in toxic activities of different proteins to different groups of organism, as previously demonstrated in scorpion venoms, is equally shared by elapid snake venoms.     

Biochemical composition, lethality and pathophysiology of venom from two cobras-- Naja naja and N. kaouthia

The cobras Naja naja and N. kaouthia are abundant in eastern and north-eastern India, accounting for maximum snakebite deaths. Here we report on variation in the composition of Naja kaouthia and N. naja venom from eastern India on corresponding differences in the severity of pathogenesis. These two venoms differ in chromatographic elution profile through Sephadex G-50 and enzyme activity, protein and carbohydrate contents associated with each fraction. The presence of greater amounts of basic phospholipase A2, L-amino acid oxidase and low molecular weight membrane active polypeptides in the N. naja venom makes it more toxic than N. kaouthia venom. A commercial polyvalent antivenom raised against N. naja venom inactivates lethality and variety of toxic effects of homologous venom more effectively than N. kaouthia venom.     

Assessment of the role of tryptophan residues in phospholipase A2 by fluorescence quenching

Tryptophan (Trp) fluorescence of two phospholipases A2 (PLA2) from Naja naja atra and Naja nigricollis snake venoms was quenched by acrylamide and iodide. Trp residues in N. naja atra PLA2 were equally accessible to acrylamide and iodide. Iodide quenching studies indicate that there are two classes of Trp fluorophores in N. nigricollis CMS-9. The accessible class consists of Trp-18 and Trp-19. Removal of the N-terminal octapeptide caused a perturbation of the micro-environment of the Trp residues in the PLA2 enzymes. The presence of a substrate lowers the susceptibility of the Trp residues to iodide quenching in N. naja atra PLA2, suggesting that all three Trp residues are at the substrate binding site, but in N. nigricollis CMS-9 Trp-18 and Trp-19 are related to substrate binding.     

Short term effects of animal venoms on the mitotic index of the duodenal mucosa of albino rats.

Short term administration of the venoms of the snakes Naja haje, Naja nigricollis, and Cerastes vipera and of the scorpion Leiurus quinquestriatus on the mitotic index of the duodenal mucosal cells of the white rat, Rattus rattus, has been studied. All the venoms increased the number of dividing cells of the duodenal mucosa significantly. Naja haje crude venom was fractionated into three fractions. Fraction I had no effect on the mitotic index whereas fractions II and III increased it significantly. Treatment of rats with Naja haje venom fractions II and III after blocking the histamine or the serotonin receptors did not affect the stimulatory action of the two venom fractions on the mitotic index, which it increased significantly. It was suggested that the venoms of Naja haje, Naja nigricollis, Cerastes vipera, and Leiurus quinquestriatus and Naja haje venom fractions possessed a mitogenic activity. Fraction II of Naja haje venom acted through both the muscarinic and adrenergic receptors while fraction III acted on the adrenergic ones.     

Comparative study of the edema-forming activity of Costa Rican snake venoms and its neutralization by a polyvalent antivenom

The edema-forming activity of eight Costa Rican crotaline snake venoms and its neutralization by a polyvalent antivenom were studied using the mouse footpad test. All of the venoms induced edema, the highest activity being present in the venoms of Bothrops lateralis and Bothrops picadoi. When experiments were performed with preincubation of venom and antivenom, neutralization of edema was poor. Moreover, it was observed that, with some venoms, edema increased when large doses of antivenom were used. This effect was also observed when some venoms were incubated with coral snake antivenom, suggesting that venoms may release some pharmacologically active component(s) from antivenom, since the latter contains traces of alpha-2 and beta globulins. Based on these findings, an alternative approach to the study of the neutralization of edema was used; in this new method, antivenom was injected i.v. before venom administration, thereby avoiding preincubation. With this technique, a much better neutralization of edema was observed, although with some venoms it was still poor. Venoms contain low molecular weight factors which induce edema, suggesting that lack of immunogenicity of some components may cause a poor neutralization. However, such components are responsible for only a minor portion of the edema induced by crude venoms. It is suggested that experiments in which venom and antivenom are preincubated preincubated in testing the neutralization of edema should be avoided, and that a more adequate approach may be an independent inoculation of venom and antivenom.     

Snake venomics and antivenomics of Protobothrops mucrosquamatus and Viridovipera stejnegeri from Taiwan: Keys to understand the variable immune response in horses

The proteomes of the venoms of the snakes Viridovipera stejnegeri and Protobothrops mucrosquamatus from Taiwan were characterized by N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of in-gel generated tryptic peptides. Proteins belonging to the following toxin classes were identified: metalloproteinase, phospholipase A(2) (PLA(2)), serine proteinase, C-type lectin-like, CRISP, l-amino acid oxidase, disintegrin, and peptides (vasoactive and inhibitors of SVMPs). Nine horses were immunized with a mixture of these venoms. All horses developed a satisfactory immune response against lethality of the venom of V. stejnegeri, whereas only three horses reached the accepted neutralizing potency against the venom of P. mucrosquamatus. Antivenoms were prepared from pools of 'good responder' (GR) and 'poor responder' (PR) horses and compared by antivenomics and neutralization tests. A similar neutralizing response was observed between the GR and PR antivenoms against the venom of V. stejnegeri, whereas antivenom from PR had a lower neutralizing activity against effects of P. mucrosquamatus venom than antivenom from GR. The low potency of the plasma of some horses against this venom is a consequence of the low immunogenicity of the neurotoxic PLA(2) trimucrotoxin. Our results provide clues for innovating the immunization scheme to generate improved antivenoms.     

Inhibition of Sendai virus by various snake venom.

Viperid, elapid and crotalid snake venoms were screened in vitro for antiviral activity against Sendai virus. The hemolysis of 10(8) human erythrocytes in 1 ml, caused by 70 HAU of Sendai virus, was abolished when the virions were pretreated with 10 ug of the viperid venom of Echis coloratus, and was considerably diminished when pretreated with 10 ug of the venom of Echis carinatus sochureki, the cobra venoms of Naja atra and Naja nigricollis nigricollis. These venoms did not affect the erythrocytes but inhibited the virions themselves irreversibly. All other examined snake venoms had low or no antiviral activity. There was no correlation between the proteolytic and the antiviral activity of the venoms.     

Cytotoxic activity of various snake venoms on melanoma, B16F10 and chondrosarcoma

Elapid, crotalid and viperid venoms were screened in vitro and in vivo for cytotoxicity towards B16F10 melanoma and chondrosarcoma cell lines. The cytotoxic activity of elapid venoms was considerably higher than that of viperid or crotalid venoms. Elapid venoms disrupted the cell membrane within the first hour, leading to cell death. The strongest activity was found in the venom of Naja nigricollis. The venoms of some Viperidae and of all Crotalidae examined caused the cells to become rounded, without loss in their original volume, and to form aggregates. These changes were reversible when cells were changed to fresh medium. In vivo experiments with the venom of Naja nigricollis were in total agreement with the results achieved in vitro with melanoma cells and the venom exhibited similar cytotoxic activity on chondrosarcoma, inhibiting its development in vivo.     

The effect of several viper venoms on prothrombin Padua.

The effect of four viper venoms (Oxyuranus scutellatus, Notechis scutatus scutatus, Echis carinatus, Naja nigricollis) on prothrombin Padua has been studied. Using Oxyuranus scutellatus venom and Notechis scutatus scutatus venom, prothrombin activity resulted to be moderately decreased similarly to what observed with other one-stage and two-stage methods. On the contrary, using Echis carinatus venom a normal level was obtained. No clotting was observed using the Naja nigricollis venom, regardless of the concentration used. The normal level of factor II obtained with Echis carinatus venom as compared with the low levels obtained with the other venoms, suggests that it acts on a different site of the prothrombin molecule.     

Snake venomics of Crotalus tigris: the minimalist toxin arsenal of the deadliest Neartic rattlesnake venom. Evolutionary Clues for generating a pan-specific antivenom against crotalid type II venoms

We report the proteomic and antivenomic characterization of Crotalus tigris venom. This venom exhibits the highest lethality for mice among rattlesnakes and the simplest toxin proteome reported to date. The venom proteome of C. tigris comprises 7-8 gene products from 6 toxin families; the presynaptic β-neurotoxic heterodimeric PLA(2), Mojave toxin, and two serine proteinases comprise, respectively, 66 and 27% of the C. tigris toxin arsenal, whereas a VEGF-like protein, a CRISP molecule, a medium-sized disintegrin, and 1-2 PIII-SVMPs each represent 0.1-5% of the total venom proteome. This toxin profile really explains the systemic neuro- and myotoxic effects observed in envenomated animals. In addition, we found that venom lethality of C. tigris and other North American rattlesnake type II venoms correlates with the concentration of Mojave toxin A-subunit, supporting the view that the neurotoxic venom phenotype of crotalid type II venoms may be described as a single-allele adaptation. Our data suggest that the evolutionary trend toward neurotoxicity, which has been also reported for the South American rattlesnakes, may have resulted by pedomorphism. The ability of an experimental antivenom to effectively immunodeplete proteins from the type II venoms of C. tigris, Crotalus horridus , Crotalus oreganus helleri, Crotalus scutulatus scutulatus, and Sistrurus catenatus catenatus indicated the feasibility of generating a pan-American anti-Crotalus type II antivenom, suggested by the identification of shared evolutionary trends among South and North American Crotalus species.     

Cross neutralization of Afro-Asian cobra and Asian krait venoms by a Thai polyvalent snake antivenom (Neuro Polyvalent Snake Antivenom)

Background

Snake envenomation is a serious public health threat in the rural areas of Asian and African countries. To date, the only proven treatment for snake envenomation is antivenom therapy. Cross-neutralization of heterologous venoms by antivenom raised against venoms of closely related species has been reported. The present study examined the cross neutralizing potential of a newly developed polyvalent antivenom, termed Neuro Polyvalent Snake Antivenom (NPAV). NPAV was produced by immunization against 4 Thai elapid venoms.

Principal Findings

In vitro neutralization study using mice showed that NPAV was able to neutralize effectively the lethality of venoms of most common Asiatic cobras (Naja spp.), Ophiophagus hannah and kraits (Bungarus spp.) from Southeast Asia, but only moderately to weakly effective against venoms of Naja from India subcontinent and Africa. Studies with several venoms showed that the in vivo neutralization potency of the NPAV was comparable to the in vitro neutralization potency. NPAV could also fully protect against N. sputatrix venom-induced cardio-respiratory depressant and neuromuscular blocking effects in anesthetized rats, demonstrating that the NPAV could neutralize most of the major lethal toxins in the Naja venom.

Conclusions/Significance

The newly developed polyvalent antivenom NPAV may find potential application in the treatment of elapid bites in Southeast Asia, especially Malaysia, a neighboring nation of Thailand. Nevertheless, the applicability of NPAV in the treatment of cobra and krait envenomations in Southeast Asian victims needs to be confirmed by clinical trials. The cross-neutralization results may contribute to the design of broad-spectrum polyvalent antivenom.     

Age-related Variation in Snake Venom： Evidence from Two Snakes （Naja atra and Deinagkistrodon acutus） in Southeastern China

In this study we explored electrophoretic profiles, enzymatic activities and immunoreactivity of neonate and adult venoms from two snakes （Naja atra and Deinagkistrodon acutus） coexisting in southeastern China. Age-related variation in electrophoretic profiles was found in both species and proteolytic and fibrinogenolytic activity was higher in neonate than adult venoms. Neonate D. acutus venom had higher 5＇ nucleotidase, PLA2, hyaluronidase and gelatinolytie activity, but lower esterolytic activity, than adult venom. Neonate and adult D. acutus venoms showed identical phosphomonoesterase, LAO and fibrinolytic activities. Neonate N. atra venom had higher phosphomonoesterase and LAO activity, but lower 5＇ nucleotidase, PLA2, hyaluronidase and Ache activities than adult venom. Neonate and adult N. atra venoms showed similar gelatinolytic activity. Further, age-dependent immunoreactivity was found in both species, and cross-reactions between homologous venoms and antiserums were closely related to venom composition. We speculate that age-related variation in venom characteristics is possibly driven by evolutionary forces associated with ontogenetic shifts in dietary habits, competition and predation pressure.     

Snake venomics and antivenomics of the arboreal neotropical pitvipers Bothriechis lateralis and Bothriechis schlegelii

We report the comparative proteomic characterization of the venoms of two related neotropical arboreal pitvipers from Costa Rica of the genus Bothriechis, B. lateralis (side-striped palm pit viper) and B. schlegelii (eyelash pit viper). The crude venoms were fractionated by reverse-phase HPLC, 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 venom proteomes of B. lateralis and B. schlegelii comprise similar number of distinct proteins belonging, respectively, to 8 and 7 protein families. The two Bothriechis venoms contain bradykinin-potentiating peptides (BPPs), and proteins from the phospholipase A 2 (PLA 2), serine proteinase, l-amino acid oxidase (LAO), cysteine-rich secretory protein (CRISP), and Zn (2+)-dependent metalloproteinase (SVMP) families, albeit each species exhibit different relative abundances. Each venom also contains unique components, for example, snake venom vascular endothelial growth factor (svVEGF) and C-type lectin-like molecules in B. lateralis, and Kazal-type serine proteinase inhibitor-like proteins in B. schlegelii. Using a similarity coefficient, we estimate that the similarity of the venom proteins between the two Bothriechis taxa may be <10%, indicating a high divergence in their venom compositions, in spite of the fact that both species have evolved to adapt to arboreal habits. The major toxin families of B. lateralis and B. schlegelii are SVMP (55% of the total venom proteins) and PLA 2 (44%), respectively. Their different venom toxin compositions provide clues for rationalizing the distinct signs of envenomation caused by B. schlegelii and B. lateralis. An antivenomic study of the immunoreactivity of the Instituto Clodomiro Picado (ICP) polyvalent antivenom toward Bothriechis venoms revealed that l-amino acid oxidase and SVMPs represent the major antigenic protein species in both venoms. Our results provide a ground for rationalizing the reported protection of the ICP polyvalent antivenom against the hemorrhagic, coagulant, defibrinating, caseinolytic and fibrin(ogen)olytic activities of Bothriechis ( schlegelii, lateralis) venoms. However, these analyses also evidenced the limited recognition capability of the polyvalent antivenom toward a number of Bothriechis venom components, predominantly BPPs, svVEGF, Kazal-type inhibitors, some PLA 2 proteins, some serine proteinases, and CRISP molecules.     

Neutralization of toxic and enzyme activities of 4 venoms from snakes of Guatemala and Honduras by the polyvalent antivenin produced in Costa Rica

We studied the ability of the polyvalent antivenom produced in Costa Rica to neutralize lethal, hemorrhagic, edema-forming, proteolytic, hemolytic, hyaluronidase and fibrinolytic activities of the venoms of Bothrops asper and B. nummifer from Honduras, and of Agkistrodon bilineatus and Crotalus durissus durissus from Guatemala. Neutralizing ability of antivenom was expressed as ED50 (effective dose 50%), defined as the antivenom/venom ratio at which the activity of the venom is reduced 50%. Antivenom is highly effective in the neutralization of lethal, hemorrhagic, hemolytic, hyaluronidase, and caseinolytic activities of B. asper, B. nummifer, and C. d. durissus venoms. In the case of B. nummifer venom, neutralization of fibrinolytic effect was only partial, whereas this activity was adequately neutralized when studying the venoms of B. asper and C. d. durissus. The venom of A. bilineatus was adequately neutralized by the antivenom, with the only exception of hemolytic effect that was reduced only partially. However, in quantitative terms, a relatively large volume of antivenom was required to neutralize some effects induced by A. bilineatus venom. Regarding edema-forming activity, antivenom neutralized efficiently the venoms of B. asper and A. bilineatus, whereas that of B. nummifer was neutralized only partially; on the other hand, edema induced by the venom of C. d. durissus was not neutralized at all. Immunochemical results indicate a close immunological relationship between venoms of B. asper, B. nummifer and C. d. durissus collected in Honduras and Guatemala with those of the same species collected in Costa Rica. Interspecies comparison, however, showed variation between venoms obtained from different species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Geographic and ontogenic variability in the venom of the neotropical rattlesnake Crotalus durissus: pathophysiological and therapeutic implications

A comparative study was performed on the venoms of adult specimens of the neotropical rattlesnake, Crotalus durissus, from Guatemala, Costa Rica, Venezuela and Brazil, together with the venom of newborn specimens of C. d. durissus from Costa Rica. Venoms from Brazil (C. d. terrificus) and from newborn specimens of C. d. durissus presented an electrophoretic pattern characterized by the predominance of bands with molecular mass of 36 and 15 kDa, whereas those of adult specimens of C. d. durissus from Guatemala and Costa Rica, and C. d. cumanensis from Venezuela, showed a conspicuous band of 62 kDa, and additional bands of 36, 29 and 15 kDa. Moreover, venoms from C. d. terrificus and C. d. cumanensis showed a prominent band of < 10 kDa that probably corresponds to crotamine, since a 'crotamine-like' activity was detected in these venoms upon intraperitoneal injection in mice. Venoms of C. d. terrificus, C. d cumanensis and newborn C. d. durissus induced higher lethal and myotoxic effects than those of adult C. d. durissus. In contrast, adult C. d. durissus and C. d. cumanensis venoms induced hemorrhage, whereas venoms of C. d. terrificus and newborn C. d. durissus lacked this effect. All venoms showed coagulant effect in plasma, the highest activity being observed in the venom of newborn C. d. durissus. An anti-crotalic antivenom produced by Instituto Butantan (Brazil), using C. d. terrificus venom as antigen, was effective in the neutralization of lethal, myotoxic and coagulant effects of all venoms studied, being ineffective in the neutralization of hemorrhagic activity of the venoms of C. d. cumanensis and C. d. durissus. On the other hand, a polyvalent antivenom produced by Instituto Clodomiro Picado (Costa Rica), using the venoms of C. d. durissus. Bothrops asper and Lachesis stenophrys as antigens, was able to neutralize lethal, myotoxic, coagulant and hemorrhagic effects of C. d. durissus venom, but was ineffective in the neutralization of lethality and myotoxicity of C. d. terrificus, C. d. cumanensis and newborn C. d. durissus venom. The high toxicity of South American and newborn C. d. durissus venoms is related to the presence of high concentrations of the neurotoxic phospholipase A2 complex 'crotoxin'. Accordingly, antivenom from Instituto Butantan has a much higher titer of anti-crotoxin antibodies than antivenom from Instituto Clodomiro Picado. Crotalus durissus represents an example of intraspecies variation in venom composition and pharmacology that has relevant pathophysiologic and therapeutic implications.     

The 1H nuclear-magnetic-resonance spectra of Neurotoxin I and cardiotoxin Vii4 from Naja mossambica mossambica.

Two toxins from the venom of Naja mossambica mossambica, neurotoxin I and cardiotoxin VII4, were investigated in aqueous solution by high-resolution 1H nuclear magnetic resonance (NMR) techniques at 360 MHz. The spectral characterization of the proteins included determination of the number of slowly exchanging amide protons which can be observed in 2H2O solution, measurement of the amide proton chemical shifts and exchange rates, characterization of the aromatic spin systems and the internal mobilities of aromatic rings, and studies of the pH dependence of the NMR spectra. For numerous resonances of labile and non-labile protons quite outstanding pH titration shifts were observed. It is suggested that these NMR parameters provide a useful basis for comparative structural studies of different proteins in the large group of homologous snake toxins. As a first application the NMR data presently available in the literature on neurotoxin II from Naja naja oxiana, toxin alpha from Naja nigricollis and erabutoxin a and b from Laticauda semifasciata have been used to compare these three proteins with neurotoxin I from Naja mossambica mossambica. This preliminary comparative study provides evidence that the same type of spatial structure prevails for these four homologous neurotoxins and that the folding of the backbone corresponds quite closely to that observed in the crystal structure of erabutoxin b. A second application is the comparison of cardiotoxin VII4 from Naja mossambica mossambica with the neurotoxins. The experimental data indicate that the folding of the polypeptide backbone is closely similar, but that the cardiotoxin molecule is markedly more flexible than the neurotoxins.     

Production of high titre antibody response against Russell's viper venom in mice immunized with ethanolic extract of fruits of Piper longum L. (Piperaceae) and piperine

Piper longum L. fruits have been traditionally used against snakebites in north-eastern and southern region of India. The aim of the study was to assess the production of antibody response against Russell's viper venom in mice after prophylactic immunization with ethanolic extract of fruits of Piper longum L. and piperine. The mice sera were tested for the presence of antibodies against Russell's viper venom by in vitro lethality neutralization assay and in vivo lethality neutralization assay. Polyvalent anti-snake venom serum (antivenom) manufactured by Haffkine Bio-Pharmaceutical Corporation Ltd. was used as standard. Further confirmation of presence of antibodies against the venom in sera of mice immunized with PLE and piperine was done using indirect enzyme-linked immunosorbent assay (ELISA) and double immunodiffusion test. Treatment with PLE-treated mice serum and piperine-treated mice serum was found to inhibit the lethal action of venom both in the in vitro lethality neutralization assay and in vivo lethality neutralization assay. ELISA testing indicated that there were significantly high (p < 0.01) levels of cross reactions between the PLE and piperine treated mice serum and the venom antigens. In double immunodiffusion test, a white band was observed between the two wells of antigen and antibodies for both the PLE-treated and piperine-treated mice serum. Thus it can be concluded that immunization with ethanolic extract of fruits of Piper longum and piperine produced a high titre antibody response against Russell's viper venom in mice. The antibodies against PLE and piperine could be useful in antivenom therapy of Russell's viper bites. PLE and piperine may also have a potential interest in view of the development of antivenom formulations used as antidote against snake bites.