Neutrophils activated by BJcuL, a C-type lectin isolated from Bothrops jararacussu venom, decrease the invasion potential of neuroblastoma SK-N-SH cells in vitro

Neuroblastoma is a pediatric tumor with a mortality rate of 40% in the most aggressive cases. Tumor microenvironment components as immune cells contribute to the tumor progression; thereby, the modulation of immune cells to a pro-inflammatory and antitumoral profile could potentialize the immunotherapy, a suggested approach for high-risk patients. Preview studies showed the antitumoral potential of BJcuL, a C- type lectin isolated from Bothrops jararacussu venom. It was able to induce immunomodulatory responses, promoting the rolling and adhesion of leukocytes and the activation of neutrophils. Methods: SK-N-SH cells were incubated with conditioned media (CM) obtained during the treatment of neutrophils with BJcuL and fMLP, a bacteria-derived peptide highly effective for activating neutrophil functions. Then we evaluated the effect of the same stimulation on the co-cultivation of neutrophils and SK-N-SH cells. Tumor cells were tested for viability, migration, and invasion potential. Results: In the viability assay, only neutrophils treated with BJcuL (24 h) and cultivated with SK-N-SH were cytotoxic. Migration of tumor cells decreased when incubated directly (p < 0.001) or indirectly (p < 0.005) with untreated neutrophils. When invasion potential was evaluated, neutrophils incubated with BJcuL reduced the total number of colonies of SK-N-SH cells following co-cultivation for 24 h (p < 0.005). Treatment with CM resulted in decreased anchorage-free survival following 24 h of treatment (p < 0.001). Conclusion: Data demonstrated that SK-N-SH cells maintain their migratory potential in the face of neutrophil modulation by BJcuL, but their invasive capacity was significantly reduced.(AU)

Anti-Trimeresurus albolabris venom IgY antibodies: preparation, purification and neutralization efficacy

Snakebite incidence in southwestern China is mainly attributed to one of the several venomous snakes found in the country, the white-lipped green pit viper Trimeresurus albolabris. Since antivenom produced from horses may cause numerous clinical side effects, the present study was conducted aiming to develop an alternative antivenom antibody (immunoglobulin Y - IgY) from leghorn chickens. Methods IgY in egg yolk from white leghorn chicken previously injected with T. albolabris venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot, and its neutralizing assay was conducted on mice. Results Chickens injected multiple times with T. albolabris venom elicited strong antibody responses, and from their egg yolk IgY was isolated and purified, which exhibited a single protein band on SDS-PAGE and two bands (about 65 and 35 kDa, respectively) under reduced conditions. Immunoblot analysis revealed that these IgY are polyclonal antibodies since they bind with most venom components. In the neutralizing assay, all mice survived while the ratios of IgY/venom reached up to 3.79 (50.0 mg/13.2 mg). Conclusions IgY antibody response was successfully conducted in white leghorn chicken injected with T. albolabrisvenom. IgY against T. albolabris venom was obtained for the first time, and it exhibited strong neutralizing potency on mice. These results may lay a foundation for the development of IgY antivenom with clinical applications in the future.

Bothrops snake venoms and their isolated toxins, an L-amino acid oxidase and a serine protease, modulate human complement system pathways

Background Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked byBothrops snakes. The present study aimed to assess whether Bothrops jararacussuand Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways.Methods Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t ½values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model.Results At the highest concentration tested (120 g/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 g/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 g/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum.Conclusion Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system byB. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system.

Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A2 from Bothrops atrox snake venom

Background Snake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. Standardization of methods for isolating bioactive molecules from snake venoms is extremely difficult due to the complex and highly variable composition of venoms, which can be influenced by factors such as age and geographic location of the specimen. Therefore, this study aimed to standardize a simple purification methodology for obtaining a P-I class metalloprotease (MP) and an acidic phospholipase A2 (PLA 2 ) from Bothrops atroxvenom, and biochemically characterize these molecules to enable future functional studies.Methods To obtain the toxins of interest, a method has been standardized using consecutive isolation steps. The purity level of the molecules was confirmed by RP-HPLC and SDS-PAGE. The enzymes were characterized by determining their molecular masses, isoelectric points, specific functional activity and partial amino acid sequencing.Results The metalloprotease presented molecular mass of 22.9 kDa and pI 7.4, with hemorrhagic and fibrin(ogen)olytic activities, and its partial amino acid sequence revealed high similarity with other P-I class metalloproteases. These results suggest that the isolated metalloprotease is Batroxase, a P-I metalloprotease previously described by our research group. The phospholipase A 2 showed molecular mass of 13.7 kDa and pI 6.5, with high phospholipase activity and similarity to other acidic PLA2 s from snake venoms. These data suggest that the acidic PLA2 is a novel enzyme from B. atrox venom, being denominated BatroxPLA 2 .Conclusions The present study successfully standardized a simple methodology to isolate the metalloprotease Batroxase and the acidic PLA 2 BatroxPLA2 from the venom of B. atrox, consisting mainly of classical chromatographic processes. These two enzymes will be used in future studies to evaluate their effects on the complement system and the inflammatory process, in addition to the thrombolytic potential of the metalloprotease.

Antitumor potential of the myotoxin BthTX-I from Bothrops jararacussu snake venom: evaluation of cell cycle alterations and death mechanisms induced in tumor cell lines

Background Phospholipases A 2 (PLA 2 s) are abundant components of snake venoms that have been extensively studied due to their pharmacological and pathophysiological effects on living organisms. This study aimed to assess the antitumor potential of BthTX-I, a basic myotoxic PLA 2isolated from Bothrops jararacussu venom, by evaluating in vitro processes of cytotoxicity, modulation of the cell cycle and induction of apoptosis in human (HL-60 and HepG2) and murine (PC-12 and B16F10) tumor cell lines. Methods The cytotoxic effects of BthTX-I were evaluated on the tumor cell lines HL-60 (promyelocytic leukemia), HepG2 (human hepatocellular carcinoma), PC-12 (murine pheochromocytoma) and B16F10 (murine melanoma) using the MTT method. Flow cytometry technique was used for the analysis of cell cycle alterations and death mechanisms (apoptosis and/or necrosis) induced in tumor cells after treatment with BthTX-I. Results It was observed that BthTX-I was cytotoxic to all evaluated tumor cell lines, reducing their viability in 40 to 50 %. The myotoxin showed modulating effects on the cell cycle of PC-12 and B16F10 cells, promoting delay in the G0/G1 phase. Additionally, flow cytometry analysis indicated cell death mainly by apoptosis. B16F10 was more susceptible to the effects of BthTX-I, with ~40 % of the cells analyzed in apoptosis, followed by HepG2 (~35 %), PC-12 (~25 %) and HL-60 (~4 %). Conclusions These results suggest that BthTX-I presents antitumor properties that may be useful for developing new therapeutic strategies against cancer.

Avaliação do efeito da crotoxina em animais portadores de encefalomielite autoimune experimental, um modelo de animais de esclerose múltipla / Evaluation of crotoxin effect in the experimental autoimune encephalomyelitis, an animal model of multiple sclerosis

A esclerose múltipla é uma doença inflamatória crônica, de origem autoimune, que acarreta diversas alterações motoras, cognitivas e sensitivas. Dentre as alterações sensitivas, a dor é um dos graves problemas que afetam pessoas portadoras desta doença, interferindo com diversos aspectos da vida do paciente. É importante ressaltar que a esclerose múltipla não tem cura, sendo que a terapêutica se concentra nas ações que retardam a progressão da doença e promovem o alívio dos sintomas, melhorando a qualidade da vida do paciente...

Multiple sclerosis is a Central Nervous System Inflamatory demyelinating disease that has as primary symptomps losses of sensory, cognitive and motor functions. Among the sensory alternations, pain is one of the major concern, afecting various aspects of the patients lives...

Antimicrobial activity of an L-amino acid oxidase isolated from Bothrops leucurus snake venom

Some snake venom proteins present enzymatic activities, such as L-amino acid oxidase (LAAO). The aim of this paper was to investigate the effect of Bothrops leucurus total venom (BleuTV) and its fraction LAAO (BleuLAAO) on bacteria, yeast, and promastigote forms of Leishmania amazonensis and Leishmania chagasi, and epimastigote forms of Trypanosoma cruzi. BleuTV was isolated with a Protein Pack 5PW® (Waters Corporation, USA), and several fractions were obtained. BleuLAAO was purified to high molecular homogeneity, and its N-terminal amino acid sequence shared a high degree of amino acid conservation with other LAAOs. BleuTV inhibited Staphylococcus aureus growth in a dose-dependent manner, with a minimum inhibitory concentration (MIC) of 25 ìg/mL, which corresponded to its minimum lethal concentration (MLC). BleuTV also inhibited the growth of promastigote forms of L. chagasi and L. amazonensis, with respective IC50 values of 1.94 ìg/mL and 5.49 ìg/mL. Furthermore, it repressed T. cruzi growth with an IC50 of 1.14 ìg/mL. However, BleuLAAO did not inhibit the growth of the microorganisms studied and was not toxic to macrophages. BleuTV had low toxicity against macrophages at the concentrations studied. In conclusion, whole venom from Bothrops leucurus inhibited the growth of some microorganisms, including S. aureus, Leishmania sp., and T. cruzi.

Comparison of wildlife and captivity rattlesnakes (Crotalus durissus terrificus) microbiota / Comparação da microbiota de cascavéis (Crotalus durissus terrificus) de vida-livre e cativeiro

The study evaluated and compared the aerobic microbiota from the oral cavity, cloaca and venom of Crotalus durissus terrificus snakes, recently caught from the wild and kept under quarantine (WQ), individual captivity (IC) and collective captivity (CC). Antimicrobial drug effectiveness on isolated agents also was assayed. From group I, II and III were isolated, respectively, 29 (63.04 percent), 38 (90.48 percent) and 21 (42.86 percent) microorganisms from the cloaca; 15 (32.61 percent), 3 (7.14 percent) and 25 (51.02 percent) microorganisms from the oral cavity; and, 2 (4.35 percent), 1 (2.38 percent) and 3 (6.12 percent) microorganisms from venom. The most frequent bacteria were Pseudomonas aeruginosa, Proteus vulgaris and Morganella morganii, with sensitivity to amikacin, gentamicin, norfloxacin, sulfazotrin and tobramycin. Snakes kept in semi-open captivity exhibited the fewest microorganisms in oral cavities, perhaps due to the environment in captivity, with different temperature gradients, running water, absence of daily handling, circulating air, possibility of moving around, daily cleaning, and sunlight access.

Este estudo avaliou e comparou a microflora aeróbica da cavidade oral, cloaca e veneno de serpentes Crotalus durissus terrificus recém-capturadas da natureza e mantidas sob quarentena (WQ), mantidas em cativeiro coletivo (CC) e em cativeiro individual (IC). A eficácia de drogas antimicrobianas de agentes isolados foi também avaliada. Foram isolados microorganismos dos grupos I, II e III respectivamente: 29 (63.04 por cento), 38 (90.48 por cento) e 21 (42.86 por cento) da cloaca; 15 (32.61 por cento), 3 (7.14 por cento) e 25 (51.02 por cento) da cavidade oral, e finalmente 2 (4.35 por cento), 1 (2.38 por cento) e 3 (6.12 por cento) do veneno. As bactérias mais frequentes foram Pseudomonas aeruginosa, Proteus vulgaris e Morganella morganii, com sensibilidade para amikacina, gentamicina, norfloxacina, sulfazotrina e tobramicina. Serpentes mantidas no cativeiro semi-aberto mostraram menor número de agentes infecciosos em cavidade oral, talvez devido ao ambiente de cativeiro com diferentes gradientes de temperatura, água corrente, ausência de manejo diário, ampla circulação de ar, possibilidade de movimentação pelos animais, limpeza diária e acesso ao Sol.

Purification and functional characterization of two fibrinogenolytic enzymes from Bothrops alternatus venom

Two fibrinogenolytic enzymes, Bothrops alternatus metalloprotease isoform (BaltMP)-I and II, were purified from Bothrops alternatus venom using Diethylaminoethyl (DEAE) Sephacel, Sephadex G-75 and Heparin-Agarose column chromatography. Purified BaltMP-I and II ran as single protein bands on analytical polyacrylamide gel electrophoresis and showed molecular weights of 29000 and 36000, respectively, under reducing conditions in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). BaltMP-II, but not BaltMP-I, displayed blood-clotting activity in bovine plasma, which was about 10-fold higher than that of the crude venom. Both enzymes were proteolytically active against bovine fibrinogen as substrate. When fibrinogen and each enzyme were incubated at 37°C, at a ratio of 1:100 (w/w), BaltMP-II cleaved preferentially the Aalpha -chain and more slowly the Bbeta -chain. The action of BaltMP-I was similar, but lower. None of the proteases degraded the gamma-chain of fibrinogen. The fibrinogenolytic activity of the enzymes was inhibited by 1,10-phenanthroline, suggesting they are metalloproteases. Since both enzymes were found to cause defibrinogenation when intraperitoneally (i.p.) administered to mice, they can be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.(AU)

Targeting exosites on blood coagulation proteases

A alta especificidade das proteases da coagulação tem sido atribuída não somente aos resíduos que cercam o sítio ativo, mas também a outros domínios de superfície que estão envolvidos no reconhecimento e interação com substratos macromoleculares e inibidores. Inibidores específicos da coagulação sanguínea obtidos de fontes exógenas como glândulas salivares de animais hematófagos e venenos de serpentes têm sido identificados. Alguns desses inibidores interagem com os exosítios das enzimas da coagulação. Dois exemplos são discutidos nesta curta revisão. A Botrojaracina é uma proteína derivada de veneno de serpente que se liga aos exosítios 1 e 2 da trombina. A formação do complexo impede várias atividades da trombina dependentes do exosítio 1 incluindo a clivagem do fibrinogênio e a ativação plaquetária. A Botrojaracina também interage com o proexosítio 1 da protrombina diminuindo a ativação do zimogênio pelo complexo protrombinase (FXa/FVa). O ixolaris é um inibidor com dois domínios Kunitz obtido da glândula salivar de carrapato, homólogo ao inibidor da via do fator tecidual. Recentemente foi demonstrado que o ixolaris se liga ao exosítio de ligação à heparina do FXa, impedindo o reconhecimento da protrombina pela enzima. Além disso, o ixolaris interage com o FX, possivelmente através do proexosítio de ligação à heparina. Diferentemente do FX, o complexo ixolaris-FX não é reconhecido como substrato pelo complexo tenase intrínseco (FIXa/FVIIIa). Nós concluimos que esses inibidores podem servir como ferramentas para o estudo dos exosítios da coagulação, assim como protótipos para novas drogas anticoagulantes.

Mice plasma fibrinogen consumption by thrombin-like enzyme present in rattlesnake venom from the north-east region of Argentina

Due to variability of venom components from the same species of snakes that inhabit different regions, particular properties of the venom of Crotalus durissus terrificus that inhabits the North-East of Argentina were studied. Gyroxin, a thrombin-like enzyme, was isolated from this venom by gel filtration and affinity chromatography, it was found to be homogeneous according to SDS-PAGE, with a molecular weight of 33 kDa. [quot ]Gyroxin syndrome[quot ] in mice was tested and it showed changes in the animal behavior, confirming that the isolated thrombin-like enzyme is gyroxin. Effects of this enzyme and the crude venom on mice plasmatic fibrinogen levels were determined. The mice plasma fibrinogen decreased rapidly until incoagulability during the first hour after thrombin-like enzyme injection, then reaching its normal level 10 hours after injection; whereas crude venom resulted in a 60% decrease of the mice plasma fibrinogen, reaching its normal level after the same period of time. After 1 hour of gyroxin inoculation, intravascular coagulation was observed in histological cuttings of lung, cardiac muscle and liver. The isolated enzyme showed strong hydrolyzing activity on fibrinogen and fibrin in vitro, whereas the crude venom exhibited weak hydrolyzing activity on both substrates. It is probable that this very low activity is due to the low percentage of the enzyme in the crude venom. Decreasing of plasmatic fibrinogen levels may be due to either the coagulant or hydrolyzing actions of the enzyme.

Teniendo en cuenta la variabilidadde los componentes del veneno de serpientes de una misma especie que habitan regiones diferentes, se decidióestudiar las propiedades particulares del veneno de Crotalus durissus terrificus que habita el nordeste de Argentina, Giroxina, una enzima con actividad trombínica, fue aislada del veneno por cromatografía de filtración por gel y de afinidad; se comprobó su homogeneidad y se determinó su peso molecular, 33 kDa, por SDSPAGE. Se ensayó el síndrome giroxina en ratones, los que mostraron cambios en el comportamiento, confirmandoque la enzima tipo trombina aislada es giroxina. Se evaluó la acción de esta enzima sobre los niveles de fibrinógeno plasmático en ratones, comparándola con la del veneno crudo. Se comprobó que la enzima provoca una disminución de los niveles plasmáticos de fibrinógeno hasta la incoagulabilidad, durante la primer hora de inoculación, mientras que el veneno entero produjo una reducción del nivel plasmático en un 60%; sin embargo, en ambos casos, se evidenció una rápida reposición de fibrinógeno plasmático, alcanzando sus valores normales en un plazo de 10 horas. Se observó coagulación intravascular con la administración de giroxina una hora después de la inoculación, evidenciados en estudios histológicos de tejido pulmonar, cardíaco y hepático. En ensayos realizados in vitro, la enzima aislada mostró capacidad de degradar fibrinógeno como así también coágulos de fibrina, mientras que el veneno exhibió débil actividad hidrolítica sobre ambos sustratos. Es probable que esta baja actividad sea debida a la baja concentración de la enzima en el veneno. La disminución de los niveles de fibrinógeno plasmático observado en ratones se debería a la acción coagulante de la enzima, sin embargo no se descarta que también contribuya a este proceso una acción hidrolítica sobrefibrinógeno y fibrina por parte de la enzima.

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.

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications

Intoxicación por veneno de crotalus durissus terrificus (cascabel) en ratas / Crotalus durissus terrificus (cascabel) nenom poisoning in rats

Se inocularon ratas de 180 a 220 g de peso con 120 ug/100 g de neneno desecado de Crotalus durissus terrrificus de Argentina, en 0,1 ml de soloución salina, por via i.m en grupos de 5 animales. Para la obtención de sangre y posterior sacrificio fueron anestesiados a las 3, 9 y 24 horas, tomándose muestras del músculo inoculado, del contralateral, de hígado y riñón. Se realizaron determinaciones enzimáticas y los tejidos se procesaron para histopatología. Se observó daño del tejido muscular esquelético a partir de las 3 horas, con necrosis miolítica y coagulativa. Paralelamente se pbservó un aumento plasmático de CK, AST yALT, siendo el aumento de CK máximo a las 9 horas posteriores a la inoculación. El tejido hepático se vió afectado, con congestión y dilastación de los vasos sanguíneos grandes. Los sinusoides periféricos a tales vasos presentaron hemosiderina a las 24 horas; las pequeñas vénulas contenian trombos. Los incrementos de AST y alt fueron máximos a las 24 horas. En tejido renal se observó congentión cortical a partir de 3 horas. A las 24 horas la congestión se extendió a las zonas yuxtamedulares con escasa cantidad de cilindros hialinos, Todos los animales manifestaron alteraciones neurológicasd con ptosis palpebral y midriasis bilateral, dificultad respiratoria, insensibilidad y parálisis progresiva. Se demostró que Crotalus durissus terrrificus de Argentina no solo posee actividad neurotóxica, sino también miolítica, hepatotóxica y nefrotóxica

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoeletric focusing

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7 per cent of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysls. Crotactine, a 12-kDa protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an altemative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained.

Purification, physicochemical characterization and N-terminal-amino acid sequence of a phospholipase A2 from Bothrops jararaca venom

Snake venoms usually contain multiple molecular forms of phospholipase A2 enzymes (phosphatide acyl hydrolase, E.C. 3.1.1.4; PLA2). Phospholipases A2 induce a wide range of pharmacological effects which may depend or not on the hydrolysis of phospholipids. In this study, a PLA2 from Bothrops jararaca venom was purified to homogeneity by gel filtration on a Sephacryl S-200 column, followed by FPLC reverse-phase chromatography on a Pep-RPC HR 5/5 column (yield 1.63 per cent of venom protein). The PLA2 activity of the fractions was determined by indirect hemolysis using hen's egg yolk lecithin as substrate. The enzyme is an acidic protein with PI 4.5 and an apparent molecular weight of 14,200, as estimated by gel filtration on a Superose 12 FPLC column. Similar properties have been described for PLA2 from other snake venoms. The N-terminal-sequence of the purified protein was NLMQFETMIMXXAGQ. These partial sequence data show a high degree of homology between the B. jararaca PLA2 and the enzymes from other snake venoms as well as bovine pancreatic PLA2

South American snake venom proteins antigenically related to bothrops asper myotoxins

1. the presence of proteins antigenically related to Bothrops asper myotoxins in various snake venoms, mainly from South America, was investigated by using poluclonal and monoclonal antibodies. 2. Myotoxin-like components were detected in the bothrops venoms from South america, and in the venoms of Crotalus atrox (North america), Trimerusurus flavoviridis (Japan), and Micrurus alleni (Costa Rica). 3. Cross-reactive components detected in several Bothrops venoms show a common subunit of 15-16 LDa by sodium dodcyl sulphate-polyacrylamide gel electrophoresis, although significant charge variations are evident by immunoelectrophoresis. 4. It is concluded that proteins antigenically related to B. asper nyotoxins are relatively common in the genus Bothrops and, in the light of findings discussed, are likely to posses myotoxic activity