Structural and functional caracterization of L-aminoácido oxidases isolated from snake Bothrops venoms / Caracterização funcional e estrutural de L-aminoácido oxidases isoladas de venenos de serpentes do gênero Bothrops

AUTOR(ES)

Carolina Dalaqua Sant Ana

DATA DE PUBLICAÇÃO

2008

RESUMO

The aim of this project was the isolation of three L-aminoacid oxidases from Bothrops jararaca, Bothrops moojeni and Bothrops pirajai snake venoms, respectively, in addition to their biochemical, enzymatic, pharmacological and toxicological characterization. They were named BjarLAAO-I, BmooLAAO-I and BpirLAAO-I, respectively, and their isolation involved two to three chromatographic steps. BjarLAAO-I and BpirLAAO-I required a molecular exclusion on Sephadex G-75, an affinity chromatography on Benzamidine-Sepharose and a hydrophobic step on Phenyl-Sepharose, while BmooLAAO-I was isolated through an ion-exchange on CM-Sepharose, followed by a hydrophobic chromatography on Phenyl-Sepharose. The high purity levels of these LAAOs were confirmed by SDS-PAGE and reverse phase HPLC on a C18 column. Their Mr were: 55,000, 65,000 and 66,000 for the monomers, and 110,000, 138,000 and 130,000 for the dimmers, respectively. Their pIs were 5.7, 4.7 and 4.9, respectively. This acid character was confirmed by their amino acid composition. The first 49 amino acid residues of their N-terminal region showed a high similarity among them as well as with other snake venom LAAOs. The enzymes are glycoprotein since, after deglycosilation, their Mr were reduced, although their activity upon hydrophobic and aromatic amino acids was kept. Usually, these Bothrops LAAOs did not show to be stable against heat and pH variations, their optimal temperature being around 4º, 25º and 37ºC at pH 7 to 8. The isolated LAAOs induced platelet aggregation and edema, but no hemorrhagic or myotoxic effect, even at high concentrations. Their cytotoxic activity was also evaluated on different organisms. They showed bactericidal (against E. coli and S. aureus), leishmanicidal (against L. braziliensis, L. amazonensis, L. major and L. donovani), trypanomicidal (against Trypanossoma cruzi), antitumoral (against JURKAT and SK-BR-3) and, antiviral activities (against type 3 Dengue virus). Finally, these LAAOs were able to induce apoptosis through DNA fragmentation, werefrom we verified the possible importance of H2O2 in the participation of these different pharmacological effects induced by these enzymes. The cDNA and molecular model of the BjarLAAO-I were determined and showed high similarity with molecular model of the LAAO from Calosellasma rodhostoma.

ASSUNTO(S)

antiviral activity. bothrops spp. l-amino acid oxidase venenos de serpente atividade parasiticida bothrops spp. antitumoral activity l-aminoácido oxidase snake venom atividade antitumoral e atividade antiviral. atividade bactericida bactericide activity parasiticide activity

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