Serpentes Brasileiras

Izidoro LF, Ribeiro MC, Souza GR, Sant'ana CD, Hamaguchi A, Homsi-Brandeburgo MI, Goulart LR, Beleboni RO, Nomizo A, Sampaio SV, Soares AM, Rodrigues VM.

In this work we describe the isolation of a new l-amino acid oxidase (LAAO) referred to as BpirLAAO-I from Bothrops pirajai snake venom, which was highly purified using a combination of molecular exclusion, affinity, and hydrophobic chromatography steps. BpirLAAO-I homodimeric acid glycoprotein (approximate Mr and pI of 130,000 and 4.9, respectively) displays high specificity toward hydrophobic/aromatic amino acids, while deglycosylation does not alter its enzymatic activity. The N-terminal LAAO sequence of its first 49 amino acids presented a high similarity between a amino acid sequence with other LAAOs from: Bothrops spp., Crotalus spp., Calloselasma rhodostoma, Agkistrodon spp., Trimeresurus spp., Pseudechis australis, Oxyuranus scutellatus, and Notechis scutatus. BpirLAAO-I induces time-dependent platelet aggregation, mouse paw edema, cytotoxic activity against Escherichia coli, Pseudomonas aeruginosa, Leishmania sp., and tumor cells, and also a typical fago (M13mp18) DNA fragmentation. Platelet aggregation, leishmanicidal and antitumoral activities were reduced by catalase. Thus, BpirLAAO-I is a multifunctional protein with promising biotechnological and medical applications.

Toyama MH, Toyama Dde O, Passero LF, Laurenti MD, Corbett CE, Tomokane TY, Fonseca FV, Antunes E, Joazeiro PP, Beriam LO, Martins MA, Monteiro HS, Fonteles MC.

A novel l-amino acid oxidase (LAO) (Casca LAO) from Crotalus durissus cascavella venom was purified to a high degree of molecular homogeneity using a combination of molecular exclusion and ion-exchange chromatography system. The purified monomer of LAO presented a molecular mass of 68 kDa and pI estimated in 5.43, which were determined by two-dimensional electrophoresis. The 71st N-terminal amino acid sequence of the LAO from Crotalus durissus cascavella presented a high amino acid sequence similarities with other LAOs from Colloselasma rhosostoma, Crotalus adamanteus, Agkistrodon h. blomhoffi, Agkistrodon h. halys and Trimeresurus stejnegeri. LAO displayed a Michaelis-Menten behavior with a kilometer of 46.7 microM and an optimum pH for enzymatic activity of 6.5. Casca LAO induced a dose-dependent platelet aggregation, which was abolished by catalase and inhibited by indomethacin and aspirin. These results suggest that the production of H2O2 is involved in subsequent activation of inflammatory enzymes, such as thromboxane. Casca LAO also inhibited the bacterial growth of Gram-negative (Xanthomonas axonopodis pv passiflorae) and Gram-positive (S. mutans) strains. Electron microscopy assessments of both bacterial strains suggest that the hydrogen peroxide produced by LAO induce bacterial membrane rupture and consequently loss of cytoplasmatic content. This LAO exhibited a high antileishmanic activity against the promastigote of Leishmania amazonensis in vitro, its activity was dependent on the production of hydrogen peroxide, and the 50% inhibitory concentration was estimated in 2.39 microg/ml.

Stabeli RG, Magalhaes LM, Selistre-de-Araujo HS, Oliveira EB.

It is widely accepted that immunological cross-reactivity of snake venoms is mediated by antibodies that recognize venom components bearing either amino acid sequence homology or similar biological functions. However, here we demonstrate that polyspecific Bothrops antivenom is a source of cross-reactive antibodies that interact with venom proteins of distinctive primary structures and biological functions. The homoserine lactone derivative of the undecapeptide IQRWSLDKYAM (Ile1-Hse11), excised from the l-amino acid oxidase (LAAO) of the Bothrops moojeni venom, was the ligand of an affinity resin used to isolate specific anti-Ile1-Hse11 antibodies which were instrumental in revealing immunological cross-reactivity among unrelated venom proteins. We examined the extent of the cross-reactivity of these antibodies by probing electroblots of venoms from representative snakes of genera Bothrops, Lachesis, Crotalus and Micrurus, and by unambiguous structural characterization of the affinity-purified proteins of B. moojeni venom recovered from an agarose-anti-Ile1-Hse11 column. Our results indicate that all venoms tested had at least three reactive components toward anti-Ile1-Hse11 antibodies, among which we identified two serine proteases, one phospholipase A2 homologue, and LAAO. We hypothesize that the cross-reactivity of the anti-Ile1-Hse11 antibodies to unrelated venom proteins derives from their mechanism of antigen recognition, whereby complementarity is achieved through reciprocal conformational adaptation of the reacting molecules. Also, we believe these findings have implications both in the development of improved antivenoms and the preparation of immunochemical reagents for diagnostic and scientific investigation purposes in the field of snake venoms.

Cambraia, RS(1); Malosso, MG(1); Marcussi, S(1,2); Fernandes, VC(1); Sant`Ana, CD(1, 3); Pereira, AMS(1); Giglio, JR(2); Soares, AM(1,3)

Propriedades antiofídicas do extrato aquoso de Jacaranda decurrens

Segundo dados da Organização Mundial de Saúde (OMS), de 65 a 80 % da população dos países pobres recorrem ao uso de plantas medicinais para o tratamento de seus males. Algumas plantas brasileiras têm sido utilizadas como antiofídicas na medicina popular. Entretanto, poucas são as espécies que têm sido investigadas cientificamente, e ainda menor é o número de seus componentes ativos isolados e caracterizados estrutural e funcionalmente. As raízes da planta Jacaranda decurrens, conhecida popularmante como "Carobinha", são muito indicadas como antiinflamatório, depurativo tônico e cura de ferimentos. Este trabalho avaliou os efeitos antiofídicos do extrato aquoso de J. decurrens sobre diferentes atividades induzidas por venenos de serpentes de várias espécies. O extrato aquoso foi incubado com os venenos por 30min a 37ºC em diferentes proporções e em seguida submetidos aos ensaios. A inibição da atividade fosfolipásica (enzimática) foi medida em gel de agarose suplementado com gema de ovo e eritrócitos humanos. Amostras de venenos e extrato previamente incubadas foram submetidas à eletroforese em gel de poliacrilamida (SDS-PAGE) para observar a presença ou não de proteólise das proteínas dos venenos pela ação do extrato. A atividade hemorrágica foi realizada por injeção intra-dérmica no dorso de camundongos seguida por medida dos halos hemorrágicos (cm). A indução de edema foi realizada por injeção sub-plantar em camundongos e medida das patas, com paquímetro de baixa pressão. O extrato inibiu parcialmente a atividade fosfolipásica dos venenos das serpentes Crotalus durissus terrificus, Bothrops alternatus e B. moojeni. A hemorragia induzida por B. jararacussu, B. alternatus e B. neuwiedi foi parcialmente inibida pelo extrato na proporção de 1:50 (m/m). A SDS-PAGE mostrou a integridade das proteínas dos venenos após incubação com o extrato nesta mesma proporção. O edema induzido por diferentes venenos foi parcialmente inibido pelo extrato, nos diferentes tempos medidos e concentrações utilizadas. As plantas medicinais constituem uma rica fonte de inibidores naturais de toxinas animais e o extrato desta planta exibe eficientes inibidores de proteases e fosfolipases A2 de venenos de serpentes.