Identification of protective B-cell epitopes of Atroxlysin-I: A metalloproteinase from Bothrops atrox snake venom.

Atroxlysin-I (Atr-I) is a hemorrhagic snake venom metalloproteinase (SVMP) from Bothrops atrox venom, the snake responsible for the majority of bites in the north region of South America. SVMPs like Atr-I produce toxic effects in victims including hemorrhage, inflammation, necrosis and blood coagulation deficiency. Mapping of B-cell epitopes in SVMPs might result in the identification of non-toxic molecules capable of inducing neutralizing antibodies and improving the anti-venom therapy. Here, using the SPOT-synthesis technique we identified two epitopes located in the N-ter region of Atr-I (AtrEp1-(22)YNGNSDKIRRRIHQM(36); and AtrEp2-(55)GVEIWSNKDLINVQ(68)). Based on the sequence of AtrEp1 and AtrEp2 a third peptide named Atr-I biepitope (AtrBiEp) was designed and synthesized ((23)NGNSDKIRRRIH(34)GG(55)GVEIWSNKDLINVQ(68)). AtrBiEp was used to immunize BALB/c mice. Anti-AtrBiEp serum cross-reacted against Atr-I in western blot and was able to fully neutralize the hemorrhagic activity of Atr-I. Our results provide a rational basis for the identification of neutralizing epitopes on Atr-I snake venom toxin and show that the use of synthetic peptides could improve the generation of immuno-therapeutics.

Tumor cytotoxicity of leucurolysin-B, a P-III snake venom metalloproteinase from Bothrops leucurus

Although it has been demonstrated that venoms and toxins from some snakes are able to influence the growth of tumor cells, few antitumoral compounds from Bothrops leucurus venom have been characterized. Leucurolysin-B (leuc-B) is a metalloproteinase class P-III isolated from B. leucurus which possesses an ECD-disintegrin domain. Both ECD-disentegrin and RGD-disintegrin are able to bind to cell surface integrins and inhibit their adherence to their natural ligands. In the present study, the potential efficacy and the cytotoxic effects of leuc-B on glioblastoma, breast cancer and melanoma cell lines were analyzed. The effect of leuc-B on cancer cell survival was evaluated and its 50 percent inhibitory concentration (IC50) was determined. Morphological alterations were monitored by contrast phase and fluorescent microscopy. The results demonstrated that leuc-B has potent cytotoxic effect in a micromolar range against all evaluated cancer cell lines. Morphologically, dying cells showed fragmentation, condensation of their contents concomitant with shrinkage and appearance of vacuoles. This study reports for the first time the cytotoxic effect of leuc-B from B. leucurus snake venom on tumor cells.

Mimotopes of mutalysin-II from Lachesis muta snake venom induce hemorrhage inhibitory antibodies upon vaccination of rabbits.

Mutalysin-II (mut-II) from Lachesis muta snake venom is an endopeptidase with hemorrhagic activity. A mAb against mutalysin-II that neutralized the hemorrhagic effect was produced previously. To identify the mAb epitopes, sets of 15-mer overlapping peptides covering the mut-II amino acid sequence were synthesized using the SPOT method and tested but failed to react with the mAb. Using a phage-display approach seventeen clones reactive with mAb were identified. Additional immunoassays with the peptides and mAb identified the QCTMDQGRLRCR, TCATDQGRLRCT, HCFHDQGRVRCA, HCTMDQGRLRCR and SCMLDQGRSRCR sequences as possible epitopes. Immunization of rabbits with these peptides induced antibodies that recognize mut-II and protected against the hemorrhagic effects of Lachesis venom.

Leucurogin, a new recombinant disintegrin cloned from Bothrops leucurus (white-tailed-jararaca) with potent activity upon platelet aggregation and tumor growth.

Disintegrins and disintegrins-like proteins are able to inhibit platelet aggregation and integrin-mediated cell adhesion. The aim of this study was to produce one disintegrin-like cloned from Bothrops leucurus venom gland and to characterize it regarding biological activity. The recombinant protein was purified by one step procedure involving anion-exchange chromatography (DEAE-cellulose) and presented a molecular mass of 10.4 kDa. The purified protein was able to inhibit platelet aggregation induced by collagen (IC50 = 0.65 µM) and to inhibit growth of Ehrlich tumor implanted in mice by more than 50% after 7 days administration of 10 µg/day. No effects were observed upon adenosine 5'-diphosphate (ADP)-and arachidonic acid (AA)-induced platelet aggregation. The recombinant protein was recognized by an antibody specific for jararhagin one metalloproteinase isolated from Bothrops jararaca venom, and therefore it was named leucurogin. Anti-angiogenesis effect of leucurogin was evaluated by the sponge implant model. After 7 days administration leucurogin inhibited, in a dose dependent way, the vascularization process in the sponge. Leucurogin represents a new biotechnological tool to understand biological processes where disintegrins-like are involved and may help to characterize integrins that can be involved in development and progression of malignant cells.

Antiophidian properties of plant extracts against Lachesis muta venom

Snakebites comprise a serious health problem in several countries due to their global incidence, which exceeds 2.5 million per year, and the elevated number of victim fatalities. To counteract envenomations, antivenoms have been used regularly for more than a century. Apart from side effects including anaphylactic reactions, antivenoms are not able to efficiently neutralize local tissue damage, which contributes to increasing the severity and morbidity observed in patients. This fact, in turn, may be responsible for economic hardship, particularly in rural populations of developing countries. In the present work, we evaluated the antiophidian properties of 12 Brazilian plant extracts against the hemolytic, coagulant, hemorrhagic and proteolytic effects of Lachesis muta venom. Taken together, our data revealed that most of these aqueous products were capable of inhibiting those activities at different levels, except for Sapindus saponaria extract. In contrast, Stryphnodendron barbatiman extract completely neutralized all the analyzed biological activities. Thus, we may conclude that Brazilian flora may also be useful against L. muta accidents.

Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom.

Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca, venom was fractionated through gel exclusion chromatography. The high molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0-0.5M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using l-leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti-S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H(2)O(2) production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of l-leucine. The correlation between in vivo neutralization of lethal toxicity (ED(50)) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients (r) between ED(50) and ELISA antibody titers against B. jararaca venom and LAAO were 0.846 (p<0.001) and 0.747 (p<0.001), respectively. The hemolytic and leishmanicidal (anti-Leishmania amazonensis) activity of LAAO was also determined.

Cytotoxic, thrombolytic and edematogenic activities of leucurolysin-a, a metalloproteinase from Bothrops leucurus snake venom.

Leucurolysin-a (leuc-a), a 23 kDa non-hemorrhagic metalloproteinase, is found in venom of the viper Bothrops leucurus. Here, we examine the biological consequences of leuc-a, including thrombolytic activity, direct effects on endothelial cells in culture and edematogenic activity in vivo. We demonstrate fibrinolytic activity of leuc-a, in which the protease specifically degrades alpha, beta, and gamma-gamma chains. While not causing hemorrhaging, leuc-a does cause thrombolytic activities in whole blood clots. Endothelial cells are highly resistant to leuc-a in culture. Cell viability suffered only when cells were exposed to large quantities of the protease. Nevertheless, leuc-a induces changes in cell morphology. The impact of leuc-a on cell adhesion was confirmed by an adhesion assay, in which cell adhesion to fibronectin decreased due to leuc-a. This mild cellular impact is unlike that of crude venom, where lower concentrations triggered cell death and a greater reduction in cell adhesion. Also, leuc-a increased microvessel permeability with marked edema in mice peritoneum and foot pads. These effects are similar to those of other P-I class SVPMs. These in vivo effects were weaker when crude venom was tested. In conclusion, albeit not showing significant hemorrhagic activity, leuc-a can induce a prominent edema which appears to be significant in the local effects observed after B. leucurus venom accidents.

Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom.

Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.

Antibodies against synthetic epitopes inhibit the enzymatic activity of mutalysin II, a metalloproteinase from bushmaster snake venom.

Mutalysin II (mut-II), a 22.5kDa zinc endopeptidase isolated from bushmaster (Lachesis muta muta) snake venom, is a direct acting fibrin(ogen)olytic proteinase. It induces monoclonal and polyclonal antibodies which efficiently neutralize the hemorrhagic effect of L. muta and several Bothrops whole venoms. To characterize epitopes of protective antibodies we have used the Spot method of multiple peptide synthesis to prepare 64 overlapping dodecapeptides frameshifted by three residues, covering the complete amino acid sequence of mut-II. The rabbit anti-mut-II antibodies binding pattern to peptides revealed several continuous antigenic regions: one in the N-terminal part, two in the central region and the other in the C-terminal of mut-II. By using homology modelling, a three-dimensional model of mut-II was built which showed that epitopes are surface exposed. Anti-peptide antibodies were raised against three peptides (one representative of each epitope region) covalently coupled as a mixture to keyhole limpet hemocyanin. Purified IgG from the resulting anti- peptide antibodies cross-reacted with mut-II and induced a dose-dependent inhibition of the mut-II catalyzed proteolysis of fibrinogen.

Functional characterization and epitope analysis of a recombinant dermonecrotic protein from Loxosceles intermedia spider.

In the present study the recombinant form (recLiD1) of a dermonecrotic protein present in the Brazilian brown spider Loxosceles intermedia venom was expressed in Escherichia coli cells and purified by reversed-phase HPLC using a C8 Vydac column. About 25.8mg of purified recLiD1 was produced from a litre of bacterial culture. SDS/PAGE and immunoblot analysis of the recombinant protein revealed an apparent molecular weight of 32-35kDa. The later result was confirmed by mass spectrometry (32,758Da). recLiD1 displayed dermonecrotic and platelet aggregation activities which were qualitatively similar to that displayed by the crude L. intermedia venom. However, very low sphingomyelinase D enzymatic activity and complement-dependent haemolytic activities were observed. recLiD1 immunized BALB/c mice developed an antibody response. Anti-recLiD1 antibodies recognized L. intermedia venom in an indirect enzyme-linked immunosorbent assay (ELISA) and cross-reacted with crude venoms from L. intermedia, L. gaucho and L. laeta. An in vivo protection assay carried out 5 weeks after the end of the immunization protocol showed that 75% of the vaccinated mice could resist the challenge by 2.5LD(50) of L. intermedia venom. To characterize epitopes associated with protective antibodies, we prepare sets of immobilized synthetic 15 mer overlapping peptides covering the complete amino acid sequences of the recLiD1. Antibodies revealed one antigenic region in the N-terminal part of the toxin. The amino acid sequence of this epitope was found in several dermonecrotic proteins and some of its residues have been implicated with the active site of the toxin.

Inhibition of mutalysin II, a metalloproteinase from bushmaster snake venom by human alpha2-macroglobulin and rabbit immunoglobulin.

Mutalysin II is a 22.5-kDa zinc endopeptidase isolated from Lachesis muta muta snake venom. In order to determine whether the inhibitors human alpha2-macroglobulin (alpha2-M) and rabbit antibody to mutalysin II share a common mechanism, we have investigated the inhibition of mutalysin II by these two different glycoproteins. The proteolytic activity of mutalysin II with dimethylcasein as substrate was completely inhibited by human alpha2-M and by a purified rabbit antibody to mutalysin II. The protection of fibrin(ogen) digestion by alpha2-M was slightly better than the protection offered by the antibody. In addition, the purified antibody reacted only with the metalloproteinase in bushmaster venom, as demonstrated by immunodiffusion. SDS-PAGE analysis of reduced samples showed that the interaction of mutalysin II with alpha2-M resulted in the formation of high molecular complex ( approximately 180000) and M(r) 90000 fragments generated by the venom enzyme. Also, fragments at 85 and 23 kDa were detected under non-reducing conditions after incubation of rabbit immunoglobulin with enzyme. Proteolysis of dimethylcasein as substrate revealed that the stoichiometry of inhibition was 1.0 mol of human alpha2-M and 1.5 mol of rabbit IgG antimutalysin II per mole of enzyme. Furthermore, dimethylcasein hydrolysis indicated that several viperid snake venoms, including Bothrops atrox, B. alternatus and Trimeresurus flavoviridis cross-reacted with the specific rabbit antibody to varying degrees.

Action of metalloproteinases mutalysin I and II on several components of the hemostatic and fibrinolytic systems.

The zinc endopeptidases mutalysin I (100 kDa) and mutalysin II (22.5 kDa) have been previously isolated from bushmaster (Lachesis muta muta) snake venom. Hemorrhagic activity was observed with as little as 0.5 microg (2000 units/mg) and 17.8 microg (56.2 units/mg) for mutalysin I and II, respectively. Additionally, the proteases hydrolyse the Aalpha>Bbeta chain of fibrinogen without clot formation. The specific fibrinogenolytic activity was estimated as 5. 25 and 16.3 micromol fibrinogen/min/micromol protein for mutalysin I and II, respectively. In vitro, the enzymes act directly on fibrin and are not inhibited by serine proteinase inhibitors (SERPINS). Analysis by SDS-PAGE of fibrin hydrolysis by both enzymes showed that mutalysin II (0.22 microM) completely digested the alpha- and gamma-gamma chains and partially the beta-chain (in 120 min incubation). In contrast, mutalysin I (three fold higher concentration than mutalysin II) hydrolyzed selectively the alpha-chain of fibrin leaving the beta and gamma-gamma chains unaffected. Unlike with the plasminogen activator-based thrombolytic agents (e.g., streptokinase), mutalysins do not activate plasminogen. Neither enzyme had an effect on protein C activation. Mutalysin II does not inhibit platelet aggregation in human PRP induced by collagen or ADP. However, mutalysin I showed a selective inhibitory effect on collagen-induced aggregation of human PRP; it did not affect platelet aggregation with ADP as the agonist. The present investigation demonstrates that both native and EDTA-inactivated mutalysin I dose dependently blocked aggregation of human PRP elicited by 10 microg/mL of collagen with an IC(50) of 180 and 580 nM, respectively. These studies suggest that, in addition to the metalloprotease region of mutalysin I, the disintegrin-like domain also participates in the inhibitory effect. The proteolytic activity of mutalysin II against dimethylcasein and fibrin was completely abolished by alpha2-macroglobulin (alpha2-M). The stoichiometry of inhibition was 1.0 mol of enzyme per mol of alpha2-M. In contrast, the proteolytic effect of mutalysin I against the same substrates was not significantly inhibited by alpha2-M. Therefore, the data explain why mutalysin I contributes significantly not only to local but also to systemic bleeding associated with the observed pathological effects of the venom.

Isolation of a proteinase with plasminogen-activating activity from Lachesis muta muta (bushmaster) snake venom.

A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity using gel filtration and anion exchange chromatography. SDS-PAGE under reducing conditions showed a single protein band with an Mr of 33,000 Da. It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular complex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at the scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmin-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that of the crude venom from which it was prepared. In vitro, fibrin hydrolysis using LV-PA as plasminogen activator displayed more similarity with the effect produced by streptokinase (SK). SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK. At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited weakly fibrinogenolytic activity. However, LV-PA is distinguished from thrombin in that it does not clot fibrinogen. After incubation of LV-PA with platelet-rich plasma, the enzyme (2 microM) showed no effect on platelet aggregation induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed that LV-PA exhibits a high degree of sequence identity with the TsVPA from Trimeresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85%). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators.

Neutralization of the hemorrhagic activity of Bothrops and Lachesis snake venoms by a monoclonal antibody against mutalysin-II.

One mAb reactive with mutalysin-II, a hemorrhagic metalloproteinase isolated from Lachesis muta muta venom, was produced in mice immunized with L. m. muta venom. Indirect ELISA was employed to compare the antigenic cross-reactivity among the venoms from Bothrops snakes. The mAb anti-mutalysin-II efficiently neutralized the hemorrhagic effect of both mutalysin-II and L. m. muta crude venom. Furthermore, the mAb were cross-reactive with B. alternatus, B. atrox, B. itapetiningae, B. jararaca and B. neuwiedii and showed variable potencies in neutralizing the hemorrhagic activity of several bothropic venoms.

Chimeric fibrolase: covalent attachment of an RGD-like peptide to create a potentially more effective thrombolytic agent.

We have prepared an agent possessing both thrombolytic and antiplatelet properties, by conjugating fibrolase, a direct-acting fibrinolytic enzyme isolated from southern copperhead venom, to a peptide which inhibits platelet aggregation. Heterobifunctional coupling reagents, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or sulfosuccinimidyl 6-[alpha-methyl-alpha-(2-pyridyldithio)-toluamido]hexanoate (Sulfo-LC-SMPT), were used in a molar ratio of 10:1 (coupling agent/fibrolase). The N-hydroxy-succinimide of the coupling agent reacts with surface epsilon-amino groups of lysine residues on fibrolase and provides a dithio group that is highly reactive with small thiol compounds. The derivatives obtained in the first reaction contain approximately two moles of 2-pyridyl disulphide per mole of enzyme. These derivatives were then reacted with the free thiol group in an antiplatelet peptide at a molar ratio of 2:1 (peptide/fibrolase). The peptide-fibrolase conjugate was purified by cation exchange HPLC and analyzed by amino acid analysis. The conjugate contains one mole peptide per mole of fibrolase and retains approximately 85% fibrinolytic activity. The IC50 for inhibition of platelet aggregation in human PRP is 300 nM for the conjugate and 67 nM for the antiplatelet peptide. These results demonstrate the successful formation of a novel chimeric protein with bifunctional activity.

Characterization of a hemorrhagic factor, LHF-I, isolated from the bushmaster snake (Lachesis muta muta) venom.

Hemorrhagic factor I (LHF-I) was previously purified from the venom of the bushmaster snake (Lachesis muta muta). In terms of biochemical and immunological properties, LHF-I is a glycoprotein (mol. wt 100,000, pI 4.7) consisting of two subunits; it loses its activity following mercaptoethanol treatment. LHF-I contains 0.7 g-atom zinc and 1.2 g-atom calcium per mole protein. The hemorrhagic and the proteinase activities are inhibited by EDTA; subsequent addition of Ca2+ or Mg2+ does not reverse the EDTA-induced inhibition of the hemorrhagic activity. The metalloenzyme does not hyrolyze arginine esters and is devoid of phospholipase A2 activity. It hydrolyzes the A alpha- > B beta-chain of fibrinogen without clot formation and hydrolyzes selectively the alpha-chain of fibrin, leaving the B beta- and tau-chains unaffected. Antibodies to the hemorrhagic factor in bushmaster venom were produced by immunizing rabbits with the purified protein. The antibody was purified by protein-A affinity chromatography. This antibody was also used to screen other Crotalinae venom samples for immunologically similar epitopes by ELISA assay. The purified antibody reacted only with LHF-I and two samples of bushmaster venom from different geographical locations.

Determination of the neutralizing potency of horse antivenom against bothropic and crotalic venoms by indirect enzyme immunoassay.

The use of ELISA to determine antisnake venom potency of horse immune sera should provide benefits of costs and reproducibility compared to in vivo assays. In the present investigation we evaluated the correlation between ELISA antibody levels and in vivo neutralization assays. For the indirect ELISA method, 0.016 micrograms/well of Bothrops jararaca or Crotalus durissus terrificus venom were used to coat the plates and 100 microliters/well of each sample of antibothropic or anticrotalic venom sera were used at 1:10,000 dilution. Sheep anti-horse IgG conjugated to peroxidase was added and the substrate H2O2/o-phenylenediamine produced the color that was read at 492 nm. A correlation coefficient of r = 0.97 was found for anticrotalic venom antibodies and no significant correlation was observed for antibothropic venom sera using 16 serum samples from immunized horses. However, when three antibothropic venom sera showing high in vivo neutralization potency and low absorbance in ELISA or high absorbance values and low in vivo protection were not included in the correlation analysis the coefficient value was r = 0.88. The correlation coefficient did not improve for all 16 antibothropic sera when a partially purified Bothrops jararaca venom fraction was used to coat the ELISA plates. The results indicate that ELISA could be used to determine the neutralizing potency of anticrotalic venom sera. For the antibothropic venom sera further studies are needed.

Determination of the neutralizing potency of horse antivenom against bothropic and crotalic venoms by indirect enzyme immunoassay

The use of ELISA to determine antisnake venom potency of horse immune sera should provide benefits of cost and reproducibility compared to in vivo assays. In the present investigation we evaluated the correlation between ELISA antibody levels and in vivo neutralization assays. For the indirect ELISA method, 0.016 µg/well of Bothrops jararaca or Crotalus durissus terrificus venom were used to coat the plates and 100 µl/well of each sample of antibothropic or anticrotalic venom sera were used at 1:10,000 dilution. Sheep anti-horse IgG conjugated to peroxidase was added and the substrate H202/o-phenylenediamine produced the color that was read at 492 nm. A correlation coefficient of r=0.97 was found for anticrotalic venom antibodies and no significant correlation was observed for antibothropic venom sera using 16 serum samples from immunized horses. However, when three antibothropic venom sera showing high in vivo neutralization...

Proteolytic specificity of two hemorrhagic factors, LHF-I and LHF-II, isolated from the venom of the bushmaster snake (Lachesis muta muta).

Two hemorrhagic metalloproteinases (LHF-I and LHF-II) were previously isolated from Lachesis muta muta (bushmaster snake) venom. The proteolytic activities of these hemorrhagic factors and of the crude venom were investigated using as substrate the oxidized B-chain of bovine insulin. LHF-II cleaves the Ala14-Leu15 bond of insulin B-chain very rapidly and the Phe24-Phe25, His10-Leu11 and His5-Leu6 more slowly, whereas LHF-I hydrolyzed only the Ala14-Leu15 bond. Both hemorrhagic factors cleaved the Leu-Leu bond in the fluorogenic peptide Abz-Pro-Leu-Gly-Leu-Leu-Gly-Arg-EDDnp. When the insulin B-chain was incubated with crude venom previously treated with 2.5 mM PMSF, the Ala14-Leu15 bond was also rapidly cleaved. In addition, the hemorrhagic activity and the digestion of casein remained unaltered. Both hemorrhagic and proteolytic activities were inhibited when the crude venom was treated with EDTA, confirming that only metalloproteinases are responsible for these activities. The hydrolysis of insulin B-chain and the fluorogenic heptapeptide by these proteinases was found to be in inverse relationship to their hemorrhagic activities.

Biological activities of venoms from South American snakes.

Standard assay procedures for the characterization of snake venoms, developed by the World Health Organization (WHO) Collaborating Centre for the Control of Antivenoms (CCCA), were used to analyse 33 snake venoms including eight International Reference Venoms for the assessment of lethal, defibrinogenating, procoagulant, haemorrhagic and necrotizing properties. The International Reference Venoms were assayed as part of an International Collaborative Programme for the evaluation of Venoms and Antivenoms; the results showed a close relationship to those obtained by the CCCA. Twenty-five venoms from 13 different species of medically important snakes from South America were assayed as standardized by the WHO-CCCA. Additionally, evaluation of lethal activity by the i.p. and intra cerebroventricular routes, proteolytic effects and venom-induced edema were also determined. Venom yields from captive snakes are also presented. Among the venoms examined, from the subfamily Crotalinae, the members of the genera Bothrops and Lachesis had strong haemorrhagic, proteolytic and edema-inducing activities, whereas all Crotalus durissus species had none. The Elapinae, Micrurus frontalis showed no procoagulant, defibrinogenating, haemorrhagic, necrotizing or proteolytic activities. The results reflect differences among individual samples of the same species and of different geographical regions. The results suggest that there is little or no relationship between the properties of the different venoms and that the determination of one effect cannot predict the value of the others. Therefore, the characterization of the different activities of snake venoms is necessary if toxicity is to be properly evaluated and neutralized.