Boletim informativo de toxicologia: acidentes ofídicos / Toxicology Fact Sheet: Snakebites

Os acidentes por animais peçonhentos, especialmente os acidentes ofídicos, foram incluídos pela Organização Mundial da Saúde (OMS) na lista das doenças tropicais negligenciadas que acometem, na maioria das vezes, populações pobres que vivem em áreas rurais, devido à gravidade constituem um problema de saúde pública. O sucesso no tratamento do paciente para que os mesmos não gerem sequelas graves, podendo chegar ao óbito, requer atendimento de forma rápida, com o uso adequado do soro específico quando necessário para cada espécie de serpentes e na quantidade recomendada. Em Goiás as principais serpentes que causam acidente são dos gêneros: Bothrops (jararacas), Crotalus (cascavéis) e Micrurus (coral), são considerados acidentes graves que demandam o uso de soros antivenenos específicos

Accidents by venomous animals, especially snakebites, were included by the World Health Organization (WHO) in the list of neglected tropical diseases that affect, most of the time, poor people living in rural areas, due to the seriousness of a public health problem. . Successful treatment of the patient so that they do not generate serious sequelae, which can lead to death, requires quick care, with the appropriate use of specific serum when necessary for each species of snakes and in the recommended amount. In Goiás, the main snakes that cause accidents are of the genera: Bothrops (jararacas), Crotalus (rattlesnakes) and Micrurus (coral), are considered serious accidents that require the use of specific antivenom serums

Tracking the recruitment and evolution of snake toxins using the evolutionary context provided by the Bothrops jararaca genome.

Venom is a key adaptive innovation in snakes, and how nonvenom genes were co-opted to become part of the toxin arsenal is a significant evolutionary question. While this process has been investigated through the phylogenetic reconstruction of toxin sequences, evidence provided by the genomic context of toxin genes remains less explored. To investigate the process of toxin recruitment, we sequenced the genome of Bothrops jararaca, a clinically relevant pitviper. In addition to producing a road map with canonical structures of genes encoding 12 toxin families, we inferred most of the ancestral genes for their loci. We found evidence that 1) snake venom metalloproteinases (SVMPs) and phospholipases A2 (PLA2) have expanded in genomic proximity to their nonvenomous ancestors; 2) serine proteinases arose by co-opting a local gene that also gave rise to lizard gilatoxins and then expanded; 3) the bradykinin-potentiating peptides originated from a C-type natriuretic peptide gene backbone; and 4) VEGF-F was co-opted from a PGF-like gene and not from VEGF-A. We evaluated two scenarios for the original recruitment of nontoxin genes for snake venom: 1) in locus ancestral gene duplication and 2) in locus ancestral gene direct co-option. The first explains the origins of two important toxins (SVMP and PLA2), while the second explains the emergence of a greater number of venom components. Overall, our results support the idea of a locally assembled venom arsenal in which the most clinically relevant toxin families expanded through posterior gene duplications, regardless of whether they originated by duplication or gene co-option.

Clinical implications of differential antivenom efficacy in neutralising coagulotoxicity produced by venoms from species within the arboreal viperid snake genus Trimeresurus.

Snake envenomation globally is attributed to an ever-increasing human population encroaching into snake territories. Responsible for many bites in Asia is the widespread genus Trimeresurus. While bites lead to haemorrhage, only a few species have had their venoms examined in detail. We found that Trimeresurus venom causes haemorrhaging by cleaving fibrinogen in a pseudo-procoagulation manner to produce weak, unstable, short-lived fibrin clots ultimately resulting in an overall anticoagulant effect due to fibrinogen depletion. The monovalent antivenom 'Thai Red Cross Green Pit Viper antivenin', varied in efficacy ranging from excellent neutralisation of T. albolabris venom through to T. gumprechti and T. mcgregori being poorly neutralised and T. hageni being unrecognised by the antivenom. While the results showing excellent neutralisation of some non-T. albolabris venoms (such as T. flavomaculaturs, T. fucatus, and T. macrops) needs to be confirmed with in vivo tests, conversely the antivenom failure T. hageni, and the very poor results against T. gumprechti and T. mcgregori, despite being conducted in the ideal scenario of preincubation of antivenom:venom, indicates that the likelihood of clinically relevant cross-reactivity for these species is low (T. gumprechti and T. mcgregori) to non-existent (T. hageni). These same latter three species were also not inhibited by the serine protease inhibitor AEBSF, suggesting that the toxins leading to a coagulotoxic effect in these species are non-serine proteases while in contrast T. albolabris coagulotoxicity was completely impeded by AEBSF, and thus driven by kallikrein-type serine proteases. There was a conspicuous lack of phylogenetic pattern in venom variation, with the most potent venoms (T. albolabris and T. hageni) being distant to each other on the organismal tree, and with the three most divergent and poorly neutralised venoms (T. gumprechti, T. hageni, and T. mcgregori) were also not each others closest relatives. This reinforces the paradigm that the fundamental dynamic evolution of venom results in organismal phylogeny being a poor predictor of venom potency or antivenom efficacy. This study provides a robust investigation on the differential venom effects from a wide range of Trimeresurus species on coagulation, highlighting differential fibrinogenolytic effects, while also investigating the relative antivenom neutralisation capabilities of the widely available Thai Red Cross Green Pit Viper antivenom. These results therefore have immediate, real-world implications for patients envenomed by Trimeresurus species.

Carbon monoxide inhibits the anticoagulant activity of Mojave rattlesnake venoms type A and B.

The Mojave rattlesnake is a unique species of pit viper that expresses either a highly potent phospholipase A2 (PLA2)-dependent neurotoxin containing venom nearly devoid of fibrinogenolytic metalloproteinases (venom type A) or a hemotoxic venom with a high percentage of metalloproteinases and PLA2 without any neurotoxin present (venom type B) depending on its geographical location in the Southwestern United States and Mexico. Given that PLA2 have been demonstrated to affect coagulation, it was hypothesized that the anticoagulant effects of both type A and B venoms could be assessed by thrombelastography, and determination made if these venoms were heme modulated. Both venom types were exposed to carbon monoxide releasing molecule-2 or its inactivated molecule (0 or 100 µM) in isolation and then placed in human plasma with consequent coagulation kinetics assessed by thrombelastography. It was determined that type A venom was twice as potent as an anticoagulant compared to type B venom, and that both venoms were inhibited by carbon monoxide releasing molecule-2 but not its inactivated molecule. Given the lack of proteolytic activity of type A venom and the dependence of neurotoxicity on PLA2 activity, it may be possible that carbon monoxide could inhibit neurotoxicity based on inhibition of PLA2 anticoagulant activity. These data may serve as the rationale for extension of these observations into animal models to determine if CO may inhibit not just hemotoxic venom, but also PLA2-dependent neurotoxic venom.

Extremely Divergent Haplotypes in Two Toxin Gene Complexes Encode Alternative Venom Types within Rattlesnake Species.

Natural selection is generally expected to favor one form of a given trait within a population. The presence of multiple functional variants of traits involved in activities such as feeding, reproduction, or the defense against predators is relatively uncommon within animal species. The genetic architecture and evolutionary mechanisms underlying the origin and maintenance of such polymorphisms are of special interest. Among rattlesnakes, several instances of the production of biochemically distinct neurotoxic or hemorrhagic venom types within the same species are known. Here, we investigated the genetic basis of this phenomenon in three species and found that neurotoxic and hemorrhagic individuals of the same species possess markedly different haplotypes at two toxin gene complexes. For example, neurotoxic and hemorrhagic Crotalus scutulatus individuals differ by 5 genes at the phospholipase A2 (PLA2) toxin gene complex and by 11 genes at the metalloproteinase (MP) gene complex. A similar set of extremely divergent haplotypes also underlies alternate venom types within C. helleri and C. horridus. We further show that the MP and PLA2 haplotypes of neurotoxic C. helleri appear to have been acquired through hybridization with C. scutulatus-a rare example of the horizontal transfer of a potentially highly adaptive suite of genes. These large structural variants appear analogous to immunity gene complexes in host-pathogen arms races and may reflect the impact of balancing selection at the PLA2 and MP complexes for predation on different prey.

Integrated Venomics and Venom Gland Transcriptome Analysis of Juvenile and Adult Mexican Rattlesnakes Crotalus simus, C. tzabcan, and C. culminatus Revealed miRNA-modulated Ontogenetic Shifts.

Adult rattlesnakes within genus Crotalus express one of two distinct venom phenotypes, type I (hemorrhagic) and type II (neurotoxic). In Costa Rican Central American rattlesnake, ontogenetic changes in the concentration of miRNAs modulate venom type II to type I transition. Venomics and venom gland transcriptome analyses showed that adult C. simus and C. tzabcan expressed intermediate patterns between type II and type I venoms, whereas C. culminatus had a canonical type I venom. Neonate/juvenile and adult Mexican rattlesnakes showed notable inter- and intraspecific variability in the number, type, abundance and ontogenetic shifts of the transcriptional and translational venom gland activities. These results support a role for miRNAs in the ontogenetic venom compositional changes in the three congeneric Mexican rattlesnakes. It is worth noting the finding of dual-action miRNAs, which silence the translation of neurotoxic heterodimeric PLA2 crotoxin and acidic PLA2 mRNAs while simultaneously up-regulating SVMP-targeting mRNAs. Dual transcriptional regulation potentially explains the existence of mutually exclusive crotoxin-rich (type-II) and SVMP-rich (type-I) venom phenotypic dichotomy among rattlesnakes. Our results support the hypothesis that alterations of the distribution of miRNAs, modulating the translational activity of venom gland toxin-encoding mRNAs in response to an external cue, may contribute to the mechanism generating adaptive venom variability.

The binding effectiveness of anti-r-disintegrin polyclonal antibodies against disintegrins and PII and PIII metalloproteases: An immunological survey of type A, B and A+B venoms from Mohave rattlesnakes.

Snake venoms are known to have different venom compositions and toxicity, but differences can also be found within populations of the same species contributing to the complexity of treatment of envenomated victims. One of the first well-documented intraspecies venom variations comes from the Mohave rattlesnake (Crotalus scutulatus scutulatus). Initially, three types of venoms were described; type A venom is the most toxic as a result of ~45% Mojave toxin in the venom composition, type B lacks the Mojave toxin but contains over 50% of snake venom metalloproteases (SVMPs). Also, type A+B venom contains a combination of Mojave toxin and SVMP. The use of an anti-disintegrin antibody in a simple Enzyme-Linked Immunosorbent Assay (ELISA) can be used to identify the difference between the venoms of the type A, B, and A+B Mohave rattlesnakes. This study implements the use of an anti-recombinant disintegrin polyclonal antibody (ARDPA) for the detection of disintegrins and ADAMs (a disintegrin and metalloproteases) in individual crude snake venoms of Mohave rattlesnakes (Crotalus scutulatus scutulatus) of varying geographical locations. After correlation with Western blots, coagulation activity and LD50 data, it was determined that the antibody allows for a quick and cost-efficient identification of venom types.

Canopy Venom: Proteomic Comparison among New World Arboreal Pit-Viper Venoms.

Central and South American pitvipers, belonging to the genera Bothrops and Bothriechis, have independently evolved arboreal tendencies. Little is known regarding the composition and activity of their venoms. In order to close this knowledge gap, venom proteomics and toxin activity of species of Bothriechis, and Bothrops (including Bothriopsis) were investigated through established analytical methods. A combination of proteomics and bioactivity techniques was used to demonstrate a similar diversification of venom composition between large and small species within Bothriechis and Bothriopsis. Increasing our understanding of the evolution of complex venom cocktails may facilitate future biodiscoveries.

Predicting function from sequence in a large multifunctional toxin family.

Venoms contain active substances with highly specific physiological effects and are increasingly being used as sources of novel diagnostic, research and treatment tools for human disease. Experimental characterisation of individual toxin activities is a severe rate-limiting step in the discovery process, and in-silico tools which allow function to be predicted from sequence information are essential. Toxins are typically members of large multifunctional families of structurally similar proteins that can have different biological activities, and minor sequence divergence can have significant consequences. Thus, existing predictive tools tend to have low accuracy. We investigated a classification model based on physico-chemical attributes that can easily be calculated from amino-acid sequences, using over 250 (mostly novel) viperid phospholipase A2 toxins. We also clustered proteins by sequence profiles, and carried out in-vitro tests for four major activities on a selection of isolated novel toxins, or crude venoms known to contain them. The majority of detected activities were consistent with predictions, in contrast to poor performance of a number of tested existing predictive methods. Our results provide a framework for comparison of active sites among different functional sub-groups of toxins that will allow a more targeted approach for identification of potential drug leads in the future.

Snake venomics of the pit vipers Porthidium nasutum, Porthidium ophryomegas, and Cerrophidion godmani from Costa Rica: toxicological and taxonomical insights.

Within the Neotropical pit vipers, a lineage of primarily Middle American snake species referred to as the "Porthidium group" includes the genera Atropoides, Cerrophidion, and Porthidium. In this study, the venom proteomes of Porthidium nasutum, P. ophryomegas, and Cerrophidion godmani from Costa Rica were analyzed, and correlated to their toxic and enzymatic activities. Their HPLC profiles revealed a higher similarity between the two Porthidium species than between these and C. godmani. Proteins belonging to nine (P. nasutum), eight (P. ophryomegas), and nine (C. godmani) families were identified by mass spectrometry or N-terminal sequencing. Final cataloging of proteins and their relative abundances confirmed the close relationship between venoms of P. nasutum and P. ophryomegas, departing from that of C. godmani. Since the latter species had been taxonomically classified as Porthidium godmani previously, our venomic analyses agree with its current generic status. Venoms of P. nasutum and P. ophryomegas, despite containing abundant metalloproteinases and serine proteinases, lack procoagulant activity on human plasma, in contrast to venom of C. godmani. The latter induced strong myotoxicity in mice, which correlates with its high proportion of phospholipases A(2), whereas venoms from the two Porthidium species, containing lower amounts of these enzymes, induced only mild muscle damage.

Deconstructing a complex molecular phenotype: population-level variation in individual venom proteins in Eastern Massasauga Rattlesnakes (Sistrurus c. catenatus).

Identifying the molecular basis for complex adaptations such as the toxic proteins used by venomous snakes to subdue and digest prey is an important step in understanding the evolutionary and functional basis for such traits. Recent proteomics-based analyses have made possible the identification of all constituent proteins in whole venom samples. Here we exploit this advance to study patterns of population-level variation in venom proteins from 254 adult eastern massasauga rattlesnakes (Sistrurus c. catenatus) collected from 10 populations. Analysis of presence-absence variation in specific proteins from 1D PAGE gels shows that: (1) The frequency spectra for individual protein bands is U-shaped with a large number of specific proteins either being consistently "common" or "rare" across populations possibly reflecting functional differences. (2) Multivariate axes which summarize whole venom variation consist of bands from all major types of proteins implying the integration of functionally distinct components within the overall venom phenotype. (3) There is significant differentiation in venom proteins across populations and the specific classes of proteins contributing to this differentiation have been identified. (4) Levels of population differentiation in venom proteins are not correlated with levels of neutral genetic differentiation, or genetically effective population sizes which argues that patterns of venom variation are not simply a consequence of population structure but leaves open the role of selection in generating population differences in venom. Our results identify particular classes of venom proteins and their associated genes as being fruitful targets for future studies of the molecular and functional basis for this complex adaptive phenotype.

Interspecific variation in venom composition and toxicity of Brazilian snakes from Bothrops genus.

The genus Bothrops spp. is responsible for 90% of envenomation by snakes in Brazil, and the standard treatment for snakebites is the antivenom therapy. The anti-bothropic serum produced by Butantan Institute is prepared by the hyperimmunization of horses with a pool of venoms from Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi. In this study, the biochemical and biological characteristics of the venoms from nineteen snakes of the genus Bothrops, responsible for human accidents in Brazil, were analysed. Venoms, particularly from Crotalidae and Viperidae snakes, are rich sources of serine proteases and metalloproteases and the ability of the Brazilian anti-bothropic serum to neutralize the proteolytic activity of these venoms were also tested. The results obtained here show the existence of a large range of variation in the composition and activities in Bothrops spp. toxins and demonstrate that the anti-bothropic serum is not able to fully neutralize the toxic activities of all analysed venoms. These suggest that for the preparation of a fully effective therapeutic anti-bothropic serum, other venoms should be included in the immunization mixture.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: insights for a new classification of snake venom metalloprotease subgroups.

Bothropasin is a 48kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys189 is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from PII SVMPs. The ECD motif is stabilized by the Cys277-Cys310 disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu276 of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other PIII SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other PIII members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain.

Molecular characterization and phylogenetic analysis of BjussuMP-I: a RGD-P-III class hemorrhagic metalloprotease from Bothrops jararacussu snake venom.

Snake venom metalloproteases (SVMPs) embody zinc-dependent multidomain enzymes responsible for a relevant pathophysiology in envenomation, including local and systemic hemorrhage. The molecular features responsible for hemorrhagic potency of SVMPs have been associated with their multidomains structures which can target these proteins them to several receptors of different tissues and cellular types. BjussuMP-I, a SVMP isolated from the Bothrops jararacussu venom, has been characterized as a P-III hemorrhagic metalloprotease. The complete cDNA sequence of BjussuMP-I with 1641bp encodes open reading frames of 547 amino acid residues, which conserve the common domains of P-III high molecular weight hemorrhagic metalloproteases: (i) pre-pro-peptide, (ii) metalloprotease, (iii) disintegrin-like and (iv) rich cysteine domain. BjussuMP-I induced lyses in fibrin clots and inhibited collagen- and ADP-induced platelet aggregation. We are reporting, for the first time, the primary structure of an RGD-P-III class snake venom metalloprotease. A phylogenetic analysis of the BjussuMP-I metalloprotease/catalytic domain was performed to get new insights into the molecular evolution of the metalloproteases. A theoretical molecular model of this domain was built through folding recognition (threading) techniques and refined by molecular dynamics simulation. Then, the final BjussuMP-I catalytic domain model was compared to other SVMPs and Reprolysin family proteins in order to identify eventual structural differences, which could help to understand the biochemical activities of these enzymes. The presence of large hydrophobic areas and some conserved surface charge-positive residues were identified as important features of the SVMPs and other metalloproteases.

Transcriptome analysis of Deinagkistrodon acutus venomous gland focusing on cellular structure and functional aspects using expressed sequence tags.

BACKGROUND: The snake venom gland is a specialized organ, which synthesizes and secretes the complex and abundant toxin proteins. Though gene expression in the snake venom gland has been extensively studied, the focus has been on the components of the venom. As far as the molecular mechanism of toxin secretion and metabolism is concerned, we still knew a little. Therefore, a fundamental question being arisen is what genes are expressed in the snake venom glands besides many toxin components? RESULTS: To examine extensively the transcripts expressed in the venom gland of Deinagkistrodon acutus and unveil the potential of its products on cellular structure and functional aspects, we generated 8696 expressed sequence tags (ESTs) from a non-normalized cDNA library. All ESTs were clustered into 3416 clusters, of which 40.16% of total ESTs belong to recognized toxin-coding sequences; 39.85% are similar to cellular transcripts; and 20.00% have no significant similarity to any known sequences. By analyzing cellular functional transcripts, we found high expression of some venom related genes and gland-specific genes, such as calglandulin EF-hand protein gene and protein disulfide isomerase gene. The transcripts of creatine kinase and NADH dehydrogenase were also identified at high level. Moreover, abundant cellular structural proteins similar to mammalian muscle tissues were also identified. The phylogenetic analysis of two snake venom toxin families of group III metalloproteinase and serine protease in suborder Colubroidea showed an early single recruitment event in the viperids evolutionary process. CONCLUSION: Gene cataloguing and profiling of the venom gland of Deinagkistrodon acutus is an essential requisite to provide molecular reagents for functional genomic studies needed for elucidating mechanisms of action of toxins and surveying physiological events taking place in the very specialized secretory tissue. So this study provides a first global view of the genetic programs for the venom gland of Deinagkistrodon acutus described so far and an insight into molecular mechanism of toxin secreting.

Lachesis muta (Viperidae) cDNAs reveal diverging pit viper molecules and scaffolds typical of cobra (Elapidae) venoms: implications for snake toxin repertoire evolution.

Efforts to describe toxins from the two major families of venomous snakes (Viperidae and Elapidae) usually reveal proteins belonging to few structural types, particular of each family. Here we carried on an effort to determine uncommon cDNAs that represent possible new toxins from Lachesis muta (Viperidae). In addition to nine classes of typical toxins, atypical molecules never observed in the hundreds of Viperidae snakes studied so far are highly expressed: a diverging C-type lectin that is related to Viperidae toxins but appears to be independently originated; an ohanin-like toxin, which would be the third member of the most recently described class of Elapidae toxins, related to human butyrophilin and B30.2 proteins; and a 3FTx-like toxin, a new member of the widely studied three-finger family of proteins, which includes major Elapidae neurotoxins and CD59 antigen. The presence of these common and uncommon molecules suggests that the repertoire of toxins could be more conserved between families than has been considered, and their features indicate a dynamic process of venom evolution through molecular mechanisms, such as multiple recruitments of important scaffolds and domain exchange between paralogs, always keeping a minimalist nature in most toxin structures in opposition to their nontoxin counterparts.

Cloning of two novel P-III class metalloproteinases from Trimeresurus stejnegeri venom gland.

Hemorrhagic toxins are widely distributed in viperid and crotalid snake venoms. Envenomation of Trimeresurus stejnegeri, a member of Crotalidae family, caused potent systemic and local hemorrhage. Up to now, there is no report on hemorrhage toxins from this venom. In this work, we cloned two cDNAs of P-III metalloproteinase precursors, designated as stejnihagin-A and stejnihagin-B, respectively, from T. stejnegeri venom gland. Both cDNAs encode an opening reading frame of 600 amino acid residues, containing a signal sequence, a proprotein domain, a metalloproteinase domain, a disintegrin-like domain and a cystetine-rich domain. Sequence analysis suggested that these two sequences shared highest similarity to the hemorrhagic toxin HR1b from T. flavoviridis. Aligning the deduced mature protein sequences of stejnihagin-A and stejnihagin-B with other snake venom metalloproteinases (SVMPs), we observed that stejnihagin-A and stejnihagin-B, together with HR1b shared the common cysteinyl residue at the position 100 in the metalloproteinase domain. In combination with the phylogenetic analysis, we presumed that stejnihagin-A, stejnihagin-B and HR1b might constitute a novel subclass of P-III SVMPs, named P-IIIc.

Crotacetin, a novel snake venom C-type lectin, is homolog of convulxin

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins, which are able to interfere with hemostasis. They share significant similarity in their primary structures with C-type lectins of other animals, and also present a conserved carbohydrate recognition domain (CRD). A very well studied sv C-type lectin is the heterodimeric toxin, convulxin (CVX), from the venoms of South American rattlesnakes, Crotalus durissus terrificus and C. d. cascavella. It consists of two subunits, alfa (CVXa, 13.9 kDa) and beta (CVXb, 12.6 kDa), joined by inter and intra-chain disulfide bounds, and is arranged in a tetrameric a4b4 conformation. Convulxin is able to activate platelet and induce their aggregation by acting via p62/GPVI collagen receptor. Several cDNA precursors, homolog of CVX subunits, were cloned by PCR homology screening. As determined by computational analysis, one of them, named crotacetin b subunit, was predicted as a polypeptide with a tridimensional conformation very similar to other subunits of convulxin-like snake toxins. Crotacetin was purified from C. durissus venoms by gel permeation and reverse phase high performance liquid chromatography. The heterodimeric crotacetin is expressed in the venoms of several C. durissus subspecies, but it is prevalent in the venom of C. durissus cascavella. As inferred from homology modeling, crotacetin induces platelet aggregation but noticeably exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria

Comparative proteomics and subtyping of venom phospholipases A2 and disintegrins of Protobothrops pit vipers.

To explore the venom diversity and systematics of pit vipers under the genus Protobothrops, the venom phospholipases A2 (PLA2s) of P. mangshanensis, P. elegans and P. tokarensis were purified and characterized for the first time. The results were compared with the corresponding venom data of other co-generic species including P. mucrosquamatus, P. flavoviridis and P. jerdonii. Based on sequence features at the N-terminal regions, we identified five PLA2 subtypes, i.e., the Asp49-PLA2s with N6, E6 or R6 substitution and the Lys49-PLA2. However, not all subtypes were expressed in each of the species. Venom N6-PLA2s from P. mangshanensis and P. tokarensis venom were weakly neurotoxic toward chick biventer cervicis tissue preparations. The venoms of P. tokarensis and P. flavoviridis contained identical PLA2 isoforms. In most Protobothrop disintegrins, sequences flanking the RGD-motif are conserved. Phylogenetic analyses based on amino acid sequences of both families of the acidic PLA2s and the disintegrins clarify that these species could belong to a monophyletic group.

Purification and partial characterization of phospholipases A2 from Bothrops asper (barba amarilla) snake venom from Chiriguaná (Cesar, Colombia)

Components with phospholipase A2 activity were isolated by gel filtration and cationic exchange chromatography from the venom of Bothrops asper snakes from Chiriguaná, Colombia (9°22´N; 73°37´W). Five fractions were obtained by the gel filtration, and PLA2 activity was found in fraction 3 (F3). In the cationic exchange chromatography, F3 showed eight components with PLA2 activity. Six of these components appeared as one band in polyacrylamide gel electrophoresis (SDS-PAGE). Fractions II and VII exhibited an optimal activity at pH 9 and 52ºC. The optimum calcium concentration for fraction II was 48 mM and for fraction VII, 384 mM. Both fractions showed thermal stability. Fraction II was stable at pH values between 2.5 and 9, and fraction VII, between 2.5 and 8. The Michaelis Menten constant (K M) was 3.5x10-3 M for fraction II and 1.6x10-3 M for fraction VII. The molecular weight was 16,000 Dalton for fraction II and 17,000 Dalton for fraction VII. Both isoenzymes did not show any toxic activity (DL50) at 5.3 and 4 µg/g. The two fractions showed different kinetic constant (K M), calcium requirement, and substrate specificity for haemolytic activity.