Application of proteomics techniques in the characterization of the snake venoms Bothrops insularis (Viperidae)
Bothrops insularis is an endemic snake found at ?Queimada Grande? island, located 35 km from São Paulo state coast. It does not predate on mammals, like its continental counterparts, because these are not found in the island. Instead, Bothrops insularis feeding is based on birds which could indicate a selective pressure that led to the diversity of toxins better adapted to bird hunting. The variety of biologically active proteins, enzymes and peptides found in the venom represent 90-95 % of its dry weight and is possibly responsible for all the biological effects observed in the prey. The diversity of macromolecules is, thus, mainly reported as a result of paralogue genes, which generate novel toxic activities and the diversity itself inside snake families. The proteomic techniques are excellent methodologies to access the complexity and diversity of protein mixtures, as they posses a huge power of resolution and extreme sensitivity. Thus, the objective of the present work was to apply such techniques to perform a proteomic analysis of B. insularis venom. To achieve this goal, snake venom samples were subjected to two-dimensional electrophoresis, which comprises isoelectric focusing on an immobilized pH gradient (IPG-IEF) followed by polyacrylamide gel electrophoresis (SDS-PAGE under reducing conditions). Then, the proteins were visualized by Coomassie blue staining and analyzed using the Image Master 2D program. The spots were further selected, cut, destained, hydrolyzed by trypsin treatment and the peptides were analyzed by MALDI-TOF and MALDI-TOF/TOF mass spectrometry. Our results revealed that the venom composition comprises several protein families such as metalloproteases, phospholipases, serine proteases, a platelet binding glycoprotein and a vascular endothelium growth factor. We have also attempted to partially characterize the metaloprotease isoforms and their proteolytic activity, search for the presence of glycoproteins, as well as possible degradation of venom. The data obtained by using those approaches allowed us to have an overview of this complex biological system, providing a better idea of the actual venom activity, where each enzymatic class cooperates with other proteins in a synergistic manner.
Advisor:Gilberto Barbosa Domont; Jonas Enrique Aguilar Perales
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Proteômica Bothrops insularis Proteomics Snake Venoms
Date of Publication:09/18/2006