Caracterização bioquímica e farmacológica da Sp-CTx- Toxina Multifuncional da Peçonha do Peixe Escorpião Scorpaena Plumieri

• Ano de defesa: 2017
• Autor(a) principal: Malacarne, Pedro Felipe
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal do Espírito Santo; BR; Mestrado em Bioquímica e Farmacologia; Centro de Ciências da Saúde; UFES; Programa de Pós-Graduação em Bioquímica e Farmacologia
• Palavras-chave em Português: Scorpionfish; Cytolysin; Venom; Stability; Perforina; Peixe Escorpião; Citolisina; Peçonha; Estabilidade; Medicamentos; Bioquímica; Farmacologia; Farmacologia Bioquímica e Molecular; 615
• Link de acesso: http://repositorio.ufes.br/handle/10/7867

Cytolysins are substances elaborated by many different organisms that exhibit toxic activities on cells, causing its dissolution through cytolysis. Fish venom cytolysins are multifunctional proteins that display neurotoxic, cardiotoxic and inflammatory effects, described as "protein lethal factors". A pore-forming labile hemolytic cytolysin called Sp-CTx (Scorpaena plumieri - Cytolytic Toxin) (glycoprotein, 2 subunits of 65 kDa) was recently purified from the venom of the scorpionfish Scorpaena plumieri. The aim of this work was to establish optimal storage conditions to maintain the activities of this toxin and to continue its biochemical and pharmacological characterization. For this, SpV (Scorpaena plumieri Venom) and Sp-CTx were stored in different conditions (pH, temperature and in the presence of co-solutes), for different periods of time. Afterwards, their hemolytic activity on rabbit erythrocytes was compared to the activity of the freshly extracted venom and purified toxin. We also evaluated the effects of different cations and membrane lipids on the hemolytic activity of Sp-CTx. It was demonstrated that storage of Sp-CTx at -4 ºC in pH 7,4 with 500mM NaCl, 1M (NH4)2SO4, 60mM of threalose and 10% glycerol, and at -80 and -196 ºC with glycerol 10% were effective in maintaining the hemolytic activity. Furthermore, the hemolytic activity is calcium dependent and completely inhibited by EDTA. Cholesterol and phosphatidylglycerol reduced the hemolytic activity of Sp-CTx which suggests that it is a barrel-pore-forming toxin and that the pore formation is dependent of electrostatic interactions with membrane lipids. Through western blot, using the “DIG Glycan Differentiation” kit, sugar motifs of Sp-CTx were evaluated, which suggested the presence of: galactose (1-4) N-acetylglucosamine and sialic-acid (2-3) galactose in N- and/or O- glycan complexes. Using the mouse hindpaw test it was shown that Sp-CTx induced intensive nociception and dose-dependent edematogenic responses in the mice footpad Sp-CTx. The local induced edema was considerably reduced by previous administration of aprotinin and HOE-140. These data suggest the involvement of the kinin– kallikrein system in the inflammatory response induced by Sp-CTx.