Purificação e caracterização biológica (estrutural, antibacteriana, antiparasitária, hemolítica e antigênica) de componentes do veneno da serpente Bothrops jararaca

• Ano de defesa: 2005
• Autor(a) principal: Paula Henriques Cruz Ciscotto
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Bioquímica e Imunologia; Bioquímica; Jararaca (Cobra) Veneno
• Link de acesso: http://hdl.handle.net/1843/BUOS-8TFQPD

Snake venoms contain many proteinaceous components which incapacitate and digest prey. Neurotoxins, cytotoxins, myotoxins, proteases and nucleases are found in varying quantities in snake venoms. The biochemical characterization of these compounds would facilitate the study of their functions and give a clue of their applicability in biotechnology. Venoms from 13 different animal species were tested using agar diffusion assay for antibacterial effects against Staphylococcus aureus. The venoms from five species of Bothrops showed strong antibacterial effects, mainly Bothrops jararaca venom. The crude venom from B. jararaca displayed antibacterial activity against various gram-negative and gram-positive bacteria. Purification of active fractions from crude venom was performed by molecular size-exclusion, ion exchange and heparin affinity chromatography. The crude venom and purificated fraction (HTP1) presented a killing effect in vitro against promastigote forms of Leishmania amazonensis and hemolytic effect in horse blood agar. Antibacterial, anti-Leishmania and hemolytic activities correlated with L- amino acid oxidase and were terminated by catalase addition. The oxidation activity of HTP1 was characterized for substrate specificity through analysis of various amino acids as substrates, with high oxidase activity against L-leucina and L-metionina. The botrocetin-like protein (P8 protein) was purified after ion exchange chromatography. The apparent molecular mass of the protein P8 was 29,5 kDa before and 15,8 kDa and 14,4 kDa after reduction. The observed molecular mass, obtained by mass spectrometry, was 30,260.87 Da. The observed amino acid sequence for the two peptide chains were DCPSGWSSYEGNCYKFFQQK (P8a) and XCPPDWSSYEGHCYRFFKEE (P8b). These two chain sequences displayed high degree of similarity with C-type lectin-like protein. An assay based on enzyme-linked immunosorbent assay (ELISA) was developed to test the potency of anti-Bothrops antivenoms, using P8 protein.