Purificação e Caracterização Química Parciais de uma enzima proteolítica do veneno de Bothrops moojeni (Caissaca)

• Autor(a) principal: Oliveira, Fábio de
• Data de Publicação: 1997
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UFU
• Texto Completo: https://repositorio.ufu.br/handle/123456789/26795; http://dx.doi.org/10.14393/ufu.di.1997.11
• topic: Venom; Veneno; Cobra; Snake; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA

Proteolytic action of snake venom proteinases has been determined on a great number of natural and synthetic substrates. A basic serine proteinase active on casein and fibrinogen was purifíed from Bothrops moojeni venom using only one step by chromatography on CM Sepharose fast flow column previously equilibrated with 0.05 M, pH 7.0 Tris-HCl 0.1 M KC1 and eluted with a convex concentration gradient (0.1 M - 0.45 M KC1) of the same buffer. The enzyme, named M003, is not hemorrhagic and presents only traces of blood- clotting activity. Synthetic chromogenic substrates (azoacasein and azoalbumin) where not hydrolyzed by M 003. On polyacrylamide gel electrophoresis at pH 4.3, M 003 presented a single protein band. On sodium dodecyl sulfate-polyaciylamide electrophoresis M 003 behave as a single- chain protein with a mol. wts of 26,600 in the presence and absence of P- mercaptoethanol and pi 7.8. The enzyme does not contain neutra) carbohydrates and its N-terminal amino acid is alanine. The amino acid composition showed 249 residues/moles a high contents hydrophilic amino acids and 14 half-cys residues, which should account for 7 dissulfide bonds. The proteinase cleaves the A-a chain faster than the B-p of bovine fibrinogen and shows no effect on the 8-chain. Both coagulant and proteolytic activities where inhibited when the M003 was treated with EDTA, P-mercaptoethanol and leupeptin. Specific esterolytic activities of M 003 on alfa-N-tosyl-1- arginine methyl ester (TAME) are 29.64 pmol min-1 mg-1. Total absorbance recovery for column was around 66%. M 003 represented 1.42% (w/w) of the initial desiccated venom