Fatores neutralizantes das atividades coagulante e hemorrágica da peçonha bruta de B.moojeni no soro da mesma espécie

Abstract

Changes in the hemostatic system, such as hemorrhages and changes in the coagulation cascade, are the main events resulting from poisoning by Bothrops snakes. Hemorrhage is caused by metalloproteases present in snake venom. Some animals have natural resistance to the harmful effects of snake venom. This resistance can be explained, in most cases, by the presence of neutralizing factors present in the serum of these animals. The work was subdivided into two chapters. The objective of chapter 1 was to identify the presence of inhibitors of coagulant and hemorrhagic activity present in the serum of the snake Bothrops moojeni. And the objective of chapter 2 was to isolate a bleeding inhibitor present in the serum of B. moojeni using a purification methodology already established for a metalloprotease inhibitor, Bj46a. In chapter 1, the serum was applied to a Q-Sepharose column (9.5 x 2.0 cm) previously balanced with 0.05M pH 8.0 Ammonium Bicarbonate buffer. Elution was carried out using the same stepwise buffer in the following concentrations (0.2 / 0.35 / 0.5M), resulting in 8 peaks. Peaks F2 (tubesl4-16) and F6 (tubes 46-51) neutralized the hemorrhagic activity caused by the raw venom (1:10 m / m) and peaks F6 and F8 (tubes 71-75) neutralized clotting activity (1 : 1 m / m). The F2 fraction (30mg) was rechromatographed in a Q-Sepharose column (9.5 x 2.0 cm), equilibrated with 0.05M pH 8.0 Ammonium Bicarbonate buffer. Elution was performed using the same buffer in a 0.2-0.5M convex gradient, 80ml mixing chamber, at a constant flow of 20ml / h, at room temperature, resulting in 3 F1Q2-F3Q2 peaks. The F2Q2 peak contains inhibitors of hemorrhagic activity and the electrophoretic profile indicated migration of bands similar to a low molecular weight metalloprotease inhibitor already described: Bj46a. In Chapter 2, B.moojeni serum was precipitated with ammonium sulfate in the saturation ranges: 0-40%; 40-60%; 60-80%; 80-100%. The 40-60% fraction was applied to a Hitrap Phenyl FíP column (1.6 x 2.5 cm) previously balanced with 0.01M sodium phosphate buffer containing 1M ammonium sulfate pH 7.0 Elution was performed by establishing a saline gradient linear decreasing to 100% sodium phosphate 0.1M pH 7.0 resulting in 7 pools: Bml-Bm7. The electrophoretic profile indicated the presence of bands around 55kDa, similar to Bj46a in the Bm2 pool. Bm2 was subjected to fractionationXI in HPLC column C4, reverse phase, resulting in a main peak, identified as Bml. The results of mass spectrometry and amino acid sequencing of 14 residues confirm the presence of the metalloprotease inhibitor.