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Photobiomodulation using light-emitting diode (LED) treatment has analgesic and anti-inflammatory effects which can be an effective therapeutic associated with serum therapy for local treatment of snakebites. Here we explored the effects of LED treatment on isolated macrophage under Bothrops jararacussu venom. Results showed that LED induced IL-6 and TNF-α genes down-regulation and, TGF and ARG1 genes up-regulation which indicates a polarization of macrophages to an M2 phenotype contributing to both tissue repair and resolution of inflammation.

The light-emitting diode (LED) is considered a therapeutic tool due to its anti-inflammatory, analgesic, and wound-healing effects, which occur through angiogenesis, decrease in IL-1β and IL-6 secretion, and acceleration of the cicatricial process. Snakebites are an important public health problem in tropical regions of the world. LED treatment is a therapeutic tool associated with serum therapy used to minimize the local effects of snakebites, including decrease in creatine kinase (CK) and lactate dehydrogenase (LDH) concentrations, myonecrosis, and inflammatory and haemorrhagic responses. In this study, we analysed the photobiomodulation effect of LED on the activation of murine macrophages induced by BthTX-I or BthTX-II isolated from Bothrops jararacussu venom. Photobiomodulation caused an increase in mitochondrial metabolism and a considerable decrease in cytotoxicity in murine macrophages. Moreover, it induced a decrease in reactive oxygen species and nitrogen liberation. However, photobiomodulation caused an increase in macrophage phagocytic capacity and lipid droplet formation. The results of this study corroborated with those of others in an unprecedented way and provide a better understanding of the mechanism of action of photobiomodulation, besides offering a coadjuvant action treatment for the local effects of snakebites, not achieved with serum therapy alone.

A comparative venom proteomic analysis of the Brazilian southern coral snake, M. frontalis, the Amazon coral snake M. spixii spixii, and the aquatic coral snake M. surinamensis is reported. Venoms from M. frontalis and M. s. spixii were composed mainly (>90% of the total venom proteome) by 3FTxs and PLAs in different proportions, and minor proteins from 2 to 5 protein families. Conversely, the aquatic coral snake expressed a streamlined (95%) 3FTx venom with low abundance (4.2%) of PLA molecules. A compositional-lethal activity for natural prey correlation analysis suggests that M. surinamensis venom may has evolved under strong pressure to quickly immobilize aquatic prey. On the other hand, venoms from M. frontalis and M. s. spixii, whose diet consist mainly of amphisbaenians and colubrid snakes, may have been shaped through balancing selection. Our work provides strong evidence for the occurrence in M. frontalis venom, but not in those from M. s. spixi and M. surinamensis, of a KUN-PLA complex homologue to heterodimeric venom toxins from some long-tailed monadal coral snakes that target acid-sensing receptors ASIC1a/2 evoking pain. The M. frontalis protein would represent the first example of a KUN-PLA heterodimer in a South American short-tailed triadal coral snake venom., This study was partly supported by grant BFU 2017-89103-P from the Ministerio de Ciencia, Innovación y Universidades, Madrid (Spain)to JJC. The authors wish to gratefully acknowledge the collaboration ofDr. Kayena Delaix Zaqueo inM. surinamensisvenom extraction. Authorsalso express their gratitude to Conselho Nacional de DesenvolvimentoCientífico e Tecnológico (CNPq/MCTIC), Coordenação deAperfeiçoamento de Pessoal de Nível Superior (CAPES/MEC) andFundação Rondônia de Amparo ao Desenvolvimento das AçõesCientíficas e Tecnológicas de Pesquisa do Estado de Rondônia(FAPERO) forfinancial support. The authors thank the Program forTechnological Development in Tools for Health-PDTIS-FIOCRUZ for theuse of its facilities.

The action of Cr-LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on NADPH oxidase activation in isolated human neutrophil function was investigated. This enzyme has an intrinsic activity of hydrogen peroxide production. Cr-LAAO, in its native form, induces the ROS production in neutrophil and migration of cytosolic NADPH oxidase components p40phox, p47phox and p67phox to the membrane, and Rac, a GTPase protein member, with the involvement of intracellular signaling mediated by phospho PKC-α. In its inactive form, iCr-LAAO does not induce NADPH oxidase activation in neutrophil showing that the intrinsic enzymatic activity does not have a role in this process, suggesting that its primary structure is essential for the cell's stimulation. Accordingly, the data showed for the first time that the Cr-LAAO has a role in NADPH oxidase complex activation triggering relevant proinflammatory events in human neutrophils.

The present work aimed to isolate a basic phospholipase A2 (PLA2) from Bothrops diporus snake venom (BdV), evaluate and compare the myotoxic and oedema-inducing activities, as well as the systemic effects caused by both the isolated PLA2 and BdV on Swiss mice. A Lys-49 PLA2 (BdipTX-I) was obtained through two chromatographic steps: an ion-exchange and a reverse phase. The local (oedema and myotoxicity) and systemic (hepatic and renal functions) effects were then assessed for BdipTX-I and BdV. Results showed that the oedema-inducing activity was significant in all tested doses (5 and 20 μg/paw) for both BdipTX-I and BdV. Myotoxicity was evaluated by the increase of serum CK, CK-MB and LDH, and results showed that BdV effect is more prominent than BdipTX-I effect. The systemic effects were evaluated by determining specific laboratory markers: AST, ALT, GGT, ALP, urea, creatinine, protein and calcium. BdipTX-I and BdV were able to induce renal changes in the experimental model, leading to proteinuria (induced both by BdipTX-I and by BdV) and uremia (induced only by BdV). Thus, it is concluded that the systemic effects of BdV and BdipTX-I occur differently.

Phospholipases A 2 (PLA 2 s) constitute a class of extensively studied toxins, isolated from snake venoms. Basic PLA 2 isoforms mediate various toxicological effects, while the acidic isoforms generally have higher enzymatic activities, but do not promote evident toxic effects. The functions of these acidic isoforms in snake venoms are still not completely understood and more studies are needed to characterize the biological functions and diversification of acidic toxins in order to justify their abundant presence in these secretions. Recently, Lomonte and collaborators demonstrated, in a proteomic and toxicological study, high concentrations of PLA 2 s in the venom of Agkistrodon piscivorus leucostoma. We have, herein, purified and characterized an acidic PLA 2 from this snake venom, denominated AplTx-I, in order to better understand its biochemical and structural characteristics, as well as its biological effects. AplTx-I was purified using two chromatographic steps, in association with enzymatic and biological assays. The acidic toxin was found to be one of the most abundant proteins in the venom of A. p. leucostoma; the protein was monomeric with a molecular mass of 13,885.8 Da, as identified by mass spectrometry ESI-TOF and electrophoresis. The toxin has similar primary and tridimensional structures to those of other acidic PLA 2 s, a theoretical and experimental isoelectric point of ≈5.12, and a calcium-dependent enzyme activity of 25.8985 nM/min/mg, with maximum values at 37 °C and pH 8.0. Despite its high enzymatic activity on synthetic substrate, AplTx-I did not induce high or significant myotoxic, coagulant, anticoagulant, edema, neuromuscular toxicity in mouse phrenic nerve-diaphragm preparations or antibacterial activities. Interestingly, AplTx-I triggered a high and selective neuromuscular toxicity in chick biventer cervicis preparations. These findings are relevant to provide a deeper understanding of the pharmacology, role and diversification of acidic phospholipase A 2 isoforms in snake venoms.

The action of LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function was investigated. Cr-LAAO showed no toxicity on neutrophils. Cr-LAAO in its native form induced the neutrophil chemotaxis, suggesting that its primary structure is essential for stimulation the cell. p38 MAPK and PI3K have a role as signaling pathways of CR-LAAO induced chemotaxis. This toxin also induced the production of hydrogen peroxide and stimulated phagocytosis in neutrophils. Furthermore, Cr-LAAO was able to stimulate neutrophils to release IL-6, IL-8, MPO, LTB4 and PGE2. Together, the data showed that the Cr-LAAO triggers relevant proinflammatory events.

The Micrurus genus is the American representative of Elapidae family. Micrurus spixii is endemic of South America and northern states of Brazil. Elapidic venoms contain neurotoxins that promote curare-mimetic neuromuscular blockage. In this study, biochemical and functional characterizations of M. spixii crude venom were performed and a new neurotoxic phospholipase A2 called MsPLA2-I was isolated. M. spixii crude venom caused severe swelling in the legs of tested mice and significant release of creatine kinase (CK) showing its myotoxic activity. Leishmanicidal activity against Leishmania amazonensis (IC50 1.24 μg/mL) was also observed, along with antiplasmodial activity against Plasmodium falciparum, which are unprecedented for Micrurus venoms. MsPLA2-I with a Mr 12,809.4 Da was isolated from the crude venom of M. spixii. The N-terminal sequencing of a fragment of 60 amino acids showed 80% similarity with another PLA2 from Micrurus altirostris. This toxin and the crude venom showed phospholipase activity. In a mouse phrenic nerve-diaphragm preparation, M. spixii venom and MsPLA2-I induced the blockage of both direct and indirect twitches. While the venom presented a pronounced myotoxic activity, MsPLA2-I expressed a summation of neurotoxic activity. The results of this study make M. spixii crude venom promising compounds in the exploration of molecules with microbicidal potential.

The aim of the study was to evaluate the in vitro effects of Bothrops bilineata crude venom (BbV) on isolated human neutrophil function. We proved that BbV isn't toxic towards human neutrophils. During an incubation of human neutrophils with BbV hydrogen peroxide was produced. Moreover, BbV was able to stimulate neutrophil release of proinflammatory mediators such as IL-8 and IL-6 as well as PGE2 and NETs liberation. There is no data in the literature showing the effect of BbV on the production of IL-6 and IL-8 or NETs liberation by isolated human neutrophils. Taken together our results testify that BbV triggers relevant proinflammatory events in human neutrophils.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:30:41Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:30:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-09-15 Lys49-phospholipases A2 (Lys49-PLA2s) are proteins found in bothropic snake venoms (Viperidae family) and belong to a class of proteins which presents a phospholipase A2 scaffold but are catalytically inactive. These proteins (also known as PLA2s-like toxins) exert a pronounced local myotoxic effect and are not neutralized by antivenom, being their study relevant in terms of medical and scientific interest. Despite of the several studies reported in the literature for this class of proteins only a partial consensus has been achieved concerning their functional-structural relationships. In this work, we present a comprehensive structural and functional study with the MjTX-II, a dimeric Lys49-PLA2 from Bothrops moojeni venom which includes: (i) high-resolution crystal structure; (ii) dynamic light scattering and bioinformatics studies in order to confirm its biological assembly; (iii) myographic and electrophysiological studies and, (iv) comparative studies with other Lys49-PLA2s. These comparative analyses let us to get important insights into the role of Lys122 amino acid, previously indicated as responsible for Lys49-PLA2s catalytic inactivity and added important elements to establish the correct biological assembly for this class of proteins. Furthermore, we show two unique sequential features of MjTX-II (an amino acid insertion and a mutation) in comparison to all bothropic Lys49-PLA2s that lead to a distinct way of ligand binding at the toxin's hydrophobic channel and also, allowed the presence of an additional ligand molecule in this region. These facts suggest a possible particular mode of binding for long-chain ligands that interacts with MjTX-II hydrophobic channel, a feature that may directly affect the design of structure-based ligands for Lys49-PLA2s. © 2013 Elsevier Ltd. Depto. de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP), Botucatu, SP Depto. de Farmacologia Instituto de Biociências Universidade Estadual Paulista (UNESP), Botucatu, SP Centro de Estudos de Biomoléculas Aplicadas à Saúde, CEBio Fundação Oswaldo Cruz, FIOCRUZ RondÔnia e Departamento de Medicina Universidade Federal de RondÔnia, UNIR, Porto Velho, RO Depto. de Física e Biofísica Instituto de Biociências Universidade Estadual Paulista (UNESP), Botucatu, SP Depto. de Farmacologia Instituto de Biociências Universidade Estadual Paulista (UNESP), Botucatu, SP

Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA2 homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [3H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals.

Several sesquiterpene lactone were synthesized and their inhibitive activities on phospholipase A 2 (PLA 2 ) from Bothrops jararacussu venom were evaluated. Compounds Lac01 and Lac02 were efficient against PLA 2 edema-inducing, enzymatic and myotoxic activities and it reduces around 85% of myotoxicity and around 70% of edema-inducing activity. Lac05–Lac08 presented lower efficiency in inhibiting the biological activities studied and reduce the myotoxic and edema-inducing activities around only 15%. The enzymatic activity was significantly reduced. The values of inhibition constants ( K I ) for Lac01 and Lac02 were approximately 740 μM, and for compounds Lac05–Lac08 the inhibition constants were approximately 7.622–9.240 μM. The enzymatic kinetic studies show that the sesquiterpene lactones inhibit PLA 2 in a non-competitive manner. Some aspects of the structure–activity relationships (topologic, molecular and electronic parameters) were obtained using ab initio quantum calculations and analyzed by chemometric methods (HCA and PCA). The quantum chemistry calculations show that compounds with a higher capacity of inhibiting PLA 2 (Lac01–Lac04) present lower values of highest occupied molecular orbital (HOMO) energy and molecular volume (VOL) and bigger values of hydrophobicity (LogP). These results indicate some topologic aspects of the binding site of sesquiterpene lactone derivatives and PLA 2 .

Prof. Dr. José R. Giglio (1934–2014) made a highly significant contribution to the field of Toxinology. During 48 years devoted to research and teaching Prof Giglio published more than 160 articles, with more than 4400 citations, in international journals, trained a vast amount of graduate and undergraduate students, and developed an international network of collaborators. Throughout these years, he worked with dedication and deep commitment to science, leaving an immortalized legacy. During his professional career he contributed mostly in the isolation, and biochemical and functional characterization of various protein toxins derived from animal venoms such as snakes, scorpions and spiders, in addition to his studies searching for alternative therapies for poisoning. Even after his departure, his presence and influence remains among his former students and in the outstanding legacy of his scientific contributions.

Phospholipase A 2 (PLA 2 , EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl- sn -3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A 2 from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE–Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl–Sepharose). BmooTX-I was found to be a single-chain protein of 15,000 Da and p I 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA 2 s from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-I with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA 2 s from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.

The effects of myotoxin III (MT-III), a phospholipase A2 (sPLA2) from Bothrops asper snake venom, and crotoxin B (CB), a neurotoxic and myotoxic sPLA2 from the venom of Crotalus durissus terrificus, on cyclooxygenases (COXs) expression and biosynthesis of prostaglandins (PGs) were evaluated, together with the mechanisms involved in these effects. Upon intraperitoneal injection in mice, both sPLA2s promoted the synthesis of PGD2 and PGE2, with a different time-course. MT-III, but not CB, induced COX-2 expression by peritoneal leukocytes without modification on COX-1 constitutive expression, whereas CB increased the constitutive activity of COX-1. MT-III increased the enzymatic activity of COX-1 and COX-2. Similar effects were observed when these sPLA2s were incubated with isolated macrophages, evidencing a direct effect on these inflammatory cells. Moreover, both toxins elicited the release of arachidonic acid from macrophages in vitro. Inhibition of cPLA2 by AACOCF3, but not of iPLA2 by PACOCF3 or BEL, significantly reduced PGD2, PGE2 and arachidonic acid (AA) release promoted by MT-III. These inhibitors did not affect MT-III-induced COX-2 expression. In contrast, cPLA2 inhibition did not modify the effects of CB, whereas iPLA2 inhibition reduced PGD2 and AA production induced by CB. These findings imply that distinct regulatory mechanisms leading to PGs' synthesis are triggered by these snake venom sPLA2s. Such differences are likely to explain the dissimilar patterns of inflammatory reaction elicited by these sPLA2s in vivo. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

We report here the antiproteolytic and antihemorrhagic properties of triterpenoid saponin inhibitors, named macrolobin-A and B, from Pentaclethra macroloba, against Bothrops snake venoms. The inhibitors were able to neutralize the hemorrhagic, fibrin(ogen)olytic, and proteolytic activities of class P-I and P-III metalloproteases isolated from B. neuwiedi and B. jararacussu venoms. Clotting and fibrinogenolytic activities induced by snake venoms and isolated thrombin-like enzymes were partially inhibited. Furthermore, the potential use of these inhibitors to complement antivenom therapy as an alternative treatment and/or used as molecular models for development of new therapeutical agents in the treatment of snake bite envenomations needs to be evaluated in future studies.

This article reports the purification procedure and the biochemical/functional characterization of Bp-PLA 2 , a new myotoxic acidic phospholipase A 2 from Bothrops pauloensis snake venom. It was highly purified through three chromatographic steps (ion-exchange on CM-Sepharose, hydrophobic chromatography on Phenyl-Sepharose and RP-HPLC on a C8 column). Bp-PLA 2 is a single-chain protein of 15.8 kDa and p I 4.3. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA 2 s from snake venoms. Its specific activity was 585.3 U/mg. It displayed a high indirect hemolytic activity and inhibited platelet aggregation induced by collagen or ADP. It also induced in vivo edema and myotoxicity. Pretreatment of Bp-PLA 2 with BPB reduced the enzymatic activity, the inhibitory action on platelet aggregation and myotoxicity in vitro . Morphological analyses indicated that Bp-PLA 2 induced an intense edema, with visible leukocyte infiltrate and damaged muscle cells 24 h after injection. Acidic myotoxic PLA 2 s from Bothrops snake venoms are still not extensively explored and knowledge of their structural and functional features will contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities.

Two basic myotoxic PLA(2)s, namely BnpTX-I and II, were isolated from Bothrops neuwiedi pauloensis snake venom through three chromatographic steps: ion-exchange chromatography on CM-Sepharose, gel filtration on Sephadex G-50 and reverse phase HPLC on a C18 column. Both PLA(2)s showed a M(r) around 14,000 for the monomer and 28,000 for the dimer (as estimated by SDS-PAGE), pI approximately 7.8 and approximately 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with Asp49 basic myotoxic PLA(2)s from other snake venoms. The catalytic and anticoagulant activities of BnpTX-I were higher than those of BnpTX-II. Both were able to induce cytotoxicity in vitro, as well as, myotoxicity, edema and lethality in mice. BnpTX-I also induced neurotoxic effect on mouse neuromuscular preparations and bactericidal activity on Eschericia coli and Staphylococcus aureus. After chemical modification of BnpTX-I with BPB or incubation with EDTA or Mn(2+) ions, the catalytic activity was completely abolished, while the toxic and pharmacological activities were partially reduced. Interaction with heparin inhibited the cytotoxic and bactericidal effects. Anti-BthTX-I, anti-BthTX-II and anti-115-129-C terminal antibodies strongly recognize both BnpTX-I and II. It is shown that the neurotoxic effect induced by B. neuwiedi pauloensis venom is due to the presence of myotoxic PLA(2)s. The data also corroborate the hypothesis of a partial dissociation between toxic and enzymatic domains. In addition, BnpTX-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.

A fibrinogen-clotting enzyme, Jararacussin-I, was purified from the venom of Bothrops jararacussu by a combination of ion exchange chromatography using Resource 15S resin and affinity chromatography using Benzamidine Sepharose 6B resin. Jararacussin-I displays a molecular mass of 28 kDa as estimated by sodium dodecyl sulphate–PAGE and possesses an isoelectric point of 5.0. The coagulant specific activity of the enzyme was determined to be 45.8 NIH U/mg using bovine fibrinogen as the substrate and the esterase specific activity was determined to be 258.7 U/mg. The protease inhibitors, benzamidine and DTT inhibited the esterase specific activity by 72.4 and 69.7%, respectively. The optimal temperature and pH for the degradation of both chains of fibrinogen and esterase specific activity were determined to be 37 °C and 7.4–8.0, respectively. The enzyme was inactivated at both 4 and 75 °C. Single crystals of Jararacussin-I were obtained and complete three-dimensional X-ray diffraction data was collected at the Brazilian National Synchrotron Source (LNLS) to a resolution of 2.4 A.

Aqueous extract from Casearia sylvestris leaves, a typical plant from Brazilian open pastures, was able to neutralize the hemorrhagic activity caused by Bothrops asper, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pirajai venoms. It also neutralized two hemorrhagic metalloproteinases from Bothrops asper venom. Proteolytic activity on casein induced by bothropic venoms and by isolated proteases, including Bn2 metalloproteinase from B. neuwiedi venom, was also inhibited by the C. sylvestris extract in different levels. The α-fibrinogen chain was partially protected against degradation caused by B. jararacussu venom, when this venom was incubated with C. sylvestris extract. We also observed that this extract partially increased the time of plasma coagulation caused by B. jararacussu, B. moojeni and B. neuwiedi venoms. C. sylvestris extract did not induce proteolysis in any substrate assayed.

Crotamine, a 4.88 kDa neurotoxic protein, has been purified to apparent homogeneity from Crotalus durissus terrificus venom by gel filtration on Sephadex G-75. When injected (i.p. or s.c.) in adult male Swiss mice (20–25 g), it induced a time-dose dependent analgesic effect which was inhibited by naloxone, thus suggesting an opioid action mechanism. When compared with morphine (4 mg/kg), crotamine, even in extremely low doses (133.4 μ g/kg, i.p., about 0.4% of a ld 50 ) is approximately 30-fold more potent than morphine (w/w) as an analgesic. On a molar basis it is more than 500-fold more potent than morphine. It is also much more potent than the lower molecular weight crude fractions of the same venom. The antinociceptive effects of crotamine and morphine were assayed by the hot plate test and by the acetic acid-induced writhing method. Therefore, both central and peripheral mechanisms should be involved. Histopathological analysis of the brain, liver, skeletal muscles, stomach, lungs, spleen, heart, kidneys and small intestine of the crotamine injected mice did not show any visible lesion in any of these organs by light microscopy. Since crotamine accounted for 22% (w/w) of the desiccated venom, it was identified as its major antinociceptive low molecular weight peptide component.

Large single crystals of piratoxin I, a Lys49-PLA2 homologue with low enzymatic activity, have been obtained. The crystals belong to the orthorhombic system space group P212121, and diffract X-rays to a resolution of 2.8 A. Preliminary analysis reveals the presence of two molecules in the crystallographic asymmetric unit.