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.     

Insights into the role of oligomeric state on the biological activities of crotoxin: crystal structure of a tetrameric phospholipase A2 formed by two isoforms of crotoxin B from Crotalus durissus terrificus venom

Crotoxin B (CB or Cdt PLA(2)) is a basic Asp49-PLA(2) found in the venom of Crotalus durissus terrificus and it is one of the subunits that constitute the crotoxin (Cro). This heterodimeric toxin, main component of the C. d. terrificus venom, is completed by an acidic, nontoxic, and nonenzymatic component (crotoxin A, CA or crotapotin), and it is related to important envenomation effects such as neurological disorders, myotoxicity, and renal failure. Although Cro has been crystallized since 1938, no crystal structure of this toxin or its subunits is currently available. In this work, the authors present the crystal structure of a novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2). The results suggest that these assemblies are stable in solution and show that Ser1 and Glu92 of CB1 and CB2, respectively, play an important role in the oligomerization. The tetrameric and dimeric conformations resulting from the association of the isoforms may increase the neurotoxicity of the toxin CB by the creation of new binding sites, which could improve the affinity of the molecular complexes to the presynaptic membrane.     

The protective effect exerted by ascorbic acid on DNA fragmentation of human leukocytes induced by Lachesis muta muta venom

The objective of this study was to evaluate the genotoxic and mutagenic effects of the toxins present in Lachesis muta muta's venom on human peripheral blood leukocytes and the protective potential of ascorbic acid on DNA fragmentation. The venom of L. muta muta was incubated in different concentrations (1, 2.5, 5, 7.5, 10, 15, 20, 30, 40, 50, 60, and 120 µg/mL) with human blood to evaluate DNA fragmentation using the comet, agarose gel electrophoresis, and micronucleus assays. In these concentrations evaluated, the venom of L. muta muta induced genotoxicity (comet assay and agarose gel electrophoresis) and mutagenicity (micronucleus test), but they were not cytotoxic, as they did not change the rate of cell proliferation after cytokinesis blockade with cytochalasin B. The ascorbic acid significantly inhibited the genotoxicity induced by L. muta muta venom in the proportions evaluated (1:0.1 and 1:0.5, venom/ascorbic acid - w/w). Thus, future studies are needed to elucidate the protective mechanisms of ascorbic acid on the genotoxic effects induced by toxins present in snake venoms.     

Characterization of ribonucleic acids from the venom glands of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom. Studies on template activity and base composition

RNA synthesis in the venom glands of Crotalus durissus terrificus was stimulated by the manual extraction of the venom (milking). RNA was extracted from venom glands activated by milking and fractionated by centrifugation through sucrose density gradients. Template activity for protein synthesis and base composition of the RNA fractions were studied. RNA fractions that sediment between 18S and 4S had the highest template activity. The base composition analysis indicated that the 28S and 18S rRNA have a C+G content of 65.4 and 58% respectively. The ;melting' temperature (T(m)) of DNA in 0.15m-NaCl-0.015m-trisodium citrate, pH7.0, was 85 degrees C, corresponding to a C+G content of 38%. The base ratio of the RNA fractions that showed a high template activity was intermediate between that of rRNA and homologous DNA. The possible role of these fractions in the synthesis of the two main toxins (crotoxin and crotamine) of the South American rattlesnake's venom is discussed.     

Analysis of cDNAs encoding the two subunits of crotoxin, a phospholipase A2 neurotoxin from rattlesnake venom: the acidic non enzymatic subunit derives from a phospholipase A2-like precursor

We report the sequences of three cDNAs encoding the two subunits (CA and CB) of crotoxin, a neurotoxic phospholipase A2 from the venom of the South-American rattlesnake Crotalus durissus terrificus. CB is a basic and toxic phospholipase A2 and CA is an acidic, non toxic and non enzymatic three chain containing protein which enhances the lethal potency of CB. Two cDNAs encoding precursors of CB isoforms have been isolated from a cDNA library prepared from one venom gland. Both precursors are made of the same 16 residues signal peptide followed by a polypeptide of 122 amino acid residues. The two mature sequences differ from each other at eight positions and are in good agreement with the previous polypeptide sequence reported for CB. In the case of CA, the cDNA encodes a signal peptide identical to those found in CB precursors, followed by a polypeptide of 122 amino acids clearly homologous to phospholipases A2 and including three regions which correspond to the three chains of mature CA. This demonstrates that CA is generated from a phospholipase A2-like precursor, called pro-CA, by the removal of three peptides, leaving unchanged the molecule core cross-linked by disulfide bridges. The 5'-untranslated tracts of cDNAs encoding CA and CB are nearly identical and the 3'-untranslated tracts are very similar, suggesting that the mRNAs encoding the two crotoxin subunits may result from the alternative splicing of a single gene or from the existence of a recent gene conversion. Data have been analysed in light of recent results on other phospholipases A2 from different origins.     

Biochemical,functional, structural and phylogenetic studies on Intercro,a new isoform phospholipase A2 from Crotalus durissus terrificus snake venom

Crotoxin is a neurotoxin from Crotalus durissus terrificus venom that shows immunomodulatory, anti-inflammatory, antimicrobial, antitumor and analgesic activities. Structurally, this toxin is a heterodimeric complex composed by a toxic basic PLA₂ (Crotoxin B or CB) non-covalently linked to an atoxic non-enzymatic and acidic component (Crotapotin, Crotoxin A or CA). Several CA and CB isoforms have been isolated and characterized, showing that the crotoxin venom fraction is, in fact, a mixture of different molecules derived from the combination of distinct subunit isoforms. Intercro (IC) is a protein from the same snake venom which presents high similarity in primary structure to CB, indicating that it could be an another isoform of this toxin. In this work, we compare IC to the crotoxin complex (CA/CB) and/or CB in order to understand its functional aspects. The experiments with IC revealed that it is a new toxin with different biological activities from CB, keeping its catalytic activity but presenting low myotoxicity and absence of neurotoxic activity. The results also indicated that IC is structurally similar to CB isoforms, but probably it is not able to form a neurotoxic active complex with crotoxin A as observed for CB. Moreover, structural and phylogenetic data suggest that IC is a new toxin with possible toxic effects not related to the typical CB neurotoxin.     

Intraspecific variation in the venoms of the South American rattlesnake (Crotalus durissus terrificus)

The venom of eight individual Crotalus durissus terrificus snakes from the State of Minas Gerais, Brazil, in addition to pooled venom from Butantan Institute, were compared. Snakes were captured in distinct locations, some of them 600 km apart: Conselheiro Lafaiete, Entre Rios de Minas, Itauna, Itapecerica, Lavras, Patos de Minas, Paracatu, and Santo Antonio do Amparo. The crude venoms were tested for proteolytic, phospholipase A2, platelet aggregating, and hemagglutinating activities. The venoms were also analyzed by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). Chromatographic patterns of venom proteins on both gel-filtration and anion-exchange chromatographies were also performed. All venoms presented high phospholipase A2 and platelet-aggregating activities, but only minimal hemagglutinating or proteolytic activities were found. Gel-filtration chromatography showed a characteristic profile for most venoms where four main peaks were separated, including the typical ones where convulxin and crotoxin were identified; however, peaks with high amounts of lower molecular weight proteins were found in the venoms from the Santo Antonio do Amparo location and Butantan Institute, characterizing these venoms as crotamine positive. Anion-exchange chromatographies presented a similar protein distribution pattern, although the number of peaks (up to ten) distinguished some venom samples. Consistent with these results, polyacrylamide gels that were silver stained after venom separation by PAGE or IEF presented a similar qualitative band distribution, although a quantitative heterogeneity was detected among venoms. Our results suggest that the variability found in venom components of C. d. terrificus venoms captured in Minas Gerais State may be genetically inherited and/or environmentally induced.     

Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom.

Crotamine is a component of the venom of the snake Crotalus durissus terrificus and it belongs to the myotoxin protein family. It is a 42 amino acid toxin cross-linked by three disulfide bridges and characterized by a mild toxicity (LD50 = 820 micro g per 25 g body weight, i.p. injection) when compared to other members of the same family. Nonetheless, it possesses a wide spectrum of biological functions. In fact, besides being able to specifically modify voltage-sensitive Na+ channel, it has been suggested to exhibit analgesic activity and to be myonecrotic. Here we report its solution structure determined by proton NMR spectroscopy. The secondary structure comprises a short N-terminal alpha-helix and a small antiparallel triple-stranded beta-sheet arranged in an alphabeta1beta2beta3 topology never found among toxins active on ion channels. Interestingly, some scorpion toxins characterized by a biological activity on Na+ channels similar to the one reported for crotamine, exhibit an alpha/beta fold, though with a beta1alphabeta2beta3 topology. In addition, as the antibacterial beta-defensins, crotamine interacts with lipid membranes. A comparison of crotamine with human beta-defensins shows a similar fold and a comparable net positive potential surface. To the best of our knowledge, this is the first report on the structure of a toxin from snake venom active on Na+ channel.     

Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test

The comet assay (single-cell gel electrophoresis, SCG) is being increasingly used in human biomonitoring for the detection of genotoxic exposures. Cigarette smoking is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds. Therefore, smoking should represent a relevant mutagenic exposure and lead to genotoxic effects in exposed cells. However, our previous investigations as well as several other published studies on human biomonitoring failed to show an effect of smoking on DNA migration in the comet assay, while some other studies did indicate such an effect. Although many factors can contribute to the generation of discrepant results in such studies, clear effects should be obtained after high exposure. We therefore performed a comparative study with healthy male heavy smokers (>20 cigarettes per day) and non-smokers (n=12 in each group). We measured the baseline comet assay effects in fresh whole blood samples and isolated lymphocytes. In addition, the amount of 'formamidopyrimidine DNA-glycosylase (FPG)-sensitive sites' was determined by a combination of the standard comet assay with the bacterial FPG protein. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline DNA damage was comparatively analysed. Duplicate slides from each sample were processed and analysed separately. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. Finally, to compare the comet assay results with another genetic endpoint, all blood samples were investigated in parallel by the micronucleus test (MNT). Baseline and gamma radiation-induced micronucleus frequencies were determined. None of these approaches revealed a significant difference between heavy smokers and non-smokers with regard to a genotoxic effect in peripheral blood cells.     

Anticrotalic and antitumoral activities of gel filtration fractions of aqueous extract from Tabernaemontana catharinensis (Apocynaceae)

The high mortality caused by Crotalus durissus terrificus snake venom is mainly due to crotoxin, which acts on the neuromuscular junction inhibiting the mechanism mediating acetylcholine release, thus leading to motor and respiratory paralysis and subsequently to animal death. We recently demonstrated that the aqueous extract (AE) of Tabernaemontana catharinensis can inhibit the lethal activity of C. d. terrificus venom. Eight fractions, PI to PVIII, were obtained by gel filtration of the extract on Sephadex G-10, and assayed for lethality and cytotoxicity. Fraction PVII [2.0 mg/100 g rat/50 microl saline solution (ss)] injected intramuscularly (i.m.) 20 s after the venom (240 microg) or crotoxin (200 microg/50 microl ss) neutralized the lethal activity of 2 LD50 of both. Fractions PI, PVI and PVIII (5.0 mg/100 g rat/50 microl ss) presented potent antitumoral activity in vitro against cells from human breast carcinoma (SK-BR-3) after 24 h incubation, as measured by Mosmann colorimetric method. Fraction PVII contains 12-methoxy-4-methylvoachalotine as its major component. These results demonstrate that the antivenom and antitumoral activities of the AE of T. catharinensis are exerted by different substances present in fraction PVII and fractions PI, PVI and PVIII, respectively, whose characteristics are distinct in terms of staining and Rf when analyzed by thin layer chromatography. The results also show that a preliminary fractionation by Sephadex G-10 gel filtration is a good option as a first step for isolation of biologically active substances from T. catharinensis.     

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.     

Chemical and functional homology of myotoxin a from prairie rattlesnake venom and crotamine from South American rattlesnake venom

Myonecrosis is a serious result of rattlesnake bite and constitutes a persistent clinical problem. In the current study we have isolated crotamine from the venom of Crotalus durissus terrificus to test its ability to cause structural damage to skeletal muscle, and to make direct chemical comparisons with Myotoxin a, a myotoxic polypeptide we recently isolated from prairie rattlesnake (Crotalus viridis viridis) venom. Disc gel electrophoresis, isoelectric focusing, circular dichroic spectroscopy, and amino acid analysis, all indicated a high degree of chemical similarity. Light microscope histology revealed that crotamine caused vacuolizationof skeletal muscle fibers, qualitatively the same as the vacuolization caused by Myotoxin a. The ability of these two basic snake venom polypeptides to cause structural damage to skeletal muscle fibers has significant implications toward more complete understanding of the cause of snake venom-induced myonecrosis.     

Genotoxicity and mutagenicity of Rosmarinus officinalis (Labiatae) essential oil in mammalian cells in vivo

Rosmarinus officinalis (rosemary) oil is widely used by the cosmetic, food, and pharmaceutical industries as a fragrance component of soaps, creams, lotions, and perfumes. Although it is popular, potential harmful side-effects of the oil have been described. We investigated the genotoxic and mutagenic potential of essential oil of R. officinalis in rodents, using comet, micronucleus and chromosome aberration assays. The animals were treated by gavage with one of three dosages of rosemary oil (300, 1000 or 2000 mg/kg). Liver and peripheral blood cells were collected from Swiss mice 24 h after treatment for the comet assay (genotoxicity endpoint), along with bone marrow cells for the micronucleus test (mutagenicity endpoint). Bone marrow cells were collected from Wistar rats 24 h after oil treatment for the micronucleus and chromosome aberration assays. Based on the comet assay, all three doses of rosemary oil induced significant increases in DNA damage in the mouse cells. There was a significant increase in micronucleated cells and chromosome aberrations only at the two higher doses. We conclude that rosemary essential oil provokes genotoxic and mutagenic effects when administered orally.     

Inhibition of crotoxin binding to synaptosomes by a receptor-like protein from Crotalus durissus terrificus (the South American rattlesnake)

Crotoxin (Ctx) is a potent neurotoxin of the venom of Crotalus durissus terrificus (the South American rattlesnake). Ctx is a heterodimer composed of CB, a toxic PLA(2) subunit, and CA, a non-toxic and non-enzymatic subunit, that potentiates the neurotoxicity of CB in vivo. The deleterious action of Ctx upon C. d. terrificus snakes themselves is known to be prevented by a PLA(2) inhibitor (CNF) present in their blood serum. CNF acts by replacing CA in Ctx, thus forming a new stable complex CNF-CB. This complex no longer interacts with the target receptor (TR) to deliver CB to cause its lethal effect. Furthermore, CNF-CB seems to be reminiscent of the interaction Ctx-TR at the pre-synaptic site. In the present work, the binding competition between rat brain synaptosomes (TR) and CNF for Ctx was investigated. Radiolabeled Ctx, made of CA and one isoform of CB (CA-(125)ICB(2)), was used as ligand. The competition by unlabeled Ctx was taken as a reference. The potency of CNF as a competitor was evaluated under different incubation conditions with varying time scale addition of reagents (CA-(125)ICB(2), synaptosomes and CA-CB(2) or CNF). CNF was able to inhibit the binding of the toxin to synaptosomes as well as to partially displace the toxin already bound to its membrane target. The mechanisms of competition involved were discussed and a previous schematic model of interactions between Ctx, TR and CNF was updated.     

Isolation of a crotoxin-like protein from the venom of a South American rattlesnake (Crotalus durissus collilineatus)

1. A crotoxin-like protein was isolated from the venom of a South American rattlesnake Crotalus durissus collilineatus. 2. Many of its properties are similar to those of crotoxin, including its non-covalent heterodimeric structure, electrophoretic mobility on SDS-PAGE, isoelectric focusing properties, toxicity in mice, immunological reactivity, multiple isoforms, phospholipase activity, peptide map, and instability on an anion-exchange column. 3. Results indicate that "collilineatus toxin" is strongly homologous with crotoxin, found in the venom of Crotalus durissus terrificus, and all other characterized rattlesnake neurotoxins.     

Combining phage display and molecular modeling to map the epitope of a neutralizing antitoxin antibody.

Crotoxin is a potent presynaptic neurotoxin from the venom of the rattlesnake Crotalus durissus terrificus. It is composed of the noncovalent and synergistic association of a weakly toxic phospholipase A2, CB, and a nontoxic three-chain subunit, CA, which increases the lethal potency of CB. The A-56.36 mAb is able to dissociate the crotoxin complex by binding to the CA subunit, thereby neutralizing its toxicity. Because A-56.36 and CB show sequence homology and both compete for binding to CA, we postulated that A-56.36 and CB had overlapping binding sites on CA. By screening random phage-displayed libraries with the mAb, phagotopes bearing the (D/S)GY(A/G) or AAXI consensus motifs were selected. They all bound A-56.36 in ELISA and competed with CA for mAb binding, although with different reactivities. When mice were immunized with the selected clones, polyclonal sera reacting with CA were induced. Interestingly, the raised antibodies retained the crotoxin-dissociating effect of A-56.36, suggesting that the selected peptides may be used to produce neutralizing antibodies. By combining these data with the molecular modeling of CA, it appeared that the functional epitope of A-56.36 on CA was conformational, one subregion being discontinuous and corresponding to the first family of peptides, the other subregion being continuous and composed of amino acids of the second family. Phage-displayed peptides corresponding to fragments of the two identified regions on CA reacted with A-56.36 and with CB. Our data support the hypothesis that A-56.36 and CB interact with common regions of CA, and highlight residues which are likely to be critical for CA-CB complex formation.     

Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: Assessment of geographic variation and its implication on snakebite management

We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4–115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20–25 main toxins belonging to the following protein families: disintegrin, PLA₂, serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), l-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA₂ crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA₂s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for ～ 20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted (～ 60%) recognition of PLA₂ molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA₂ molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed.     

The primary structure of crotamine (author's transl)]

The primary structure of crotamine, a basic toxin isolated from the venom of the South American rattle-snake Crotalus durissus terrificus has been determined. The polypeptide chain is composed of 42 residues of amino acids. Crotamine shows a molecular weight of 4900 and contains 6 half cystine, 9 lysine, 2 arginine, 2 histidine and 2 tryptophan residues.     

Interchain disulfide bonds in crotamine self-association

Crotamine, a basic neurotoxic protein, was isolated from the venom of the Southern Brazilian rattlesnake (Crotalus durissus terrificus) by gel filtration. The isolated protein showed a single band on PAGE at pH 4.5 and 7% (w/v) gel concentration, but two or more bands at 14% gel concentration, even in the presence of 4 M urea. After reduction and carboxymethylation, however, a single band was again detected. SDS-PAGE as well as ultracentrifugal analysis of the native (NC) and of the reduced and carboxymethylated (RCC) crotamine revealed a molecular weight of 4,500-5,000 for RCC and 9,000-10,000 for NC. Both components of a two-band crotamine preparation were isolated by preparative PAGE and characterized. Their particular electrophoretic mobility was retained. Their amino acid composition. N-terminal residue, and apparent toxicity were the same as those of the original sample. It was concluded that crotamine is able to form a dimer of 9,760 Da with two identical polypeptide chains crosslinked by interchain disulfide bonds and a shape not very far from spherical, which covalently binds extra subunits of 4,880 Da each.     

Identification of continuous interaction sites in PLA2-based protein complexes by peptide arrays

Crotoxin (CA.CB) is a β-neurotoxin from Crotalus durissus terrificus snake venom that is responsible for main envenomation effects upon biting by this snake. It is a heterodimer of an acidic protein (CA) devoid of any biological activity per se and a basic, enzymatically active, PLA₂ counterpart (CB). Both lethal and enzymatic activities of crotoxin have been shown to be inhibited by CNF, a protein from the blood of C. d. terrificus snakes. CNF replaces CA in the CA.CB complex, forming a stable, non-toxic complex CNF.CB. The molecular sites involved in the tight interfacial protein–protein interactions in these PLA₂-based complexes have not been clearly determined. To help address this question, we used the peptide arrays approach to map possible interfacial interaction sites in CA.CB and CNF.CB. Amino acid stretches putatively involved in these interactions were firstly identified in the primary structure of CB. Further analysis of the interfacial availability of these stretches in the presumed biologically active structure of CB, suggested two interaction main sites, located at the amino-terminus and β-wing regions. Peptide segments at the carboxyl-terminus of CB were also suggested to play a secondary role in the binding of both CA and CNF.