Caracterização estrutural e funcional de metaloproteases isoladas de peçonhas botrópicas

Abstract

CHAPTER II:In the present work, we demonstrate the biochemical and functional characterization of Bothropoidin, a hemorrhagic snake venom metalloproteinase isolated from Bothrops pauloensis snake venom. This protein was purified after three chromatographic steps on CM-Sepharose fast flow, HiTrep Sephacryl S-300 RP and HiTrep Capto Q. Bothropoidin was homogeneous by SDS-PAGE under reducing and nonreducing conditions, and was shown to present a single chain of 49,558 Da by MALDI TOF analysis. The protein presented isoeletric point of 3.76 and the sequence of two fragments obtained by Peptide Mass Fingerprinting (PMF) in MS (MALDI TOF\\TOF) showed significant score when compared with another SVMPs.This enzyme showed proteolytic activity upon azocasein, Aα- chain of fibrinogen, fibrin, collagen and fibronectin. The enzyme was stable at pH between 6-9 and lower temperatures when assayed on azocasein. Moreover, this activity was inhibited by EDTA, 1,10-phenanthroline and β-mercaptoethanol. This enzyme acted on blood clotting altering the aPTT and PT. Bothropoidin was able to induce hemorrhage (MHD=0.98 μg) and necrosis in the gastrocnenius muscles of mice, showing high toxicity. The protein was capable of inhibiting platelet aggregation induced by collagen and ADP and interfered with viability and cell adhesion when incubated with tEnd endothelial cells in a dose dependent manner. Taken together, these results lead us to suggest that Bothropoidin is a hemorrhagic α-fibrinogenase which can contribute significantly to the toxicity of Bothrops pauloensis envenomation. CHAPTER III: A fibrino(geno)lytic nonhemorrhagic metalloproteinase (BleucMP) was purified from Bothrops leucurus snake venom by two chromatographic steps procedure on DEAE-Sephadex A-25 followed by CM-Sepharose Fast Flow column. BleucMP represented 1.75% (w/w) of the crude venom and was homogeneous on SDS-PAGE. BleucMP analyzed by MALDI TOF/TOF, showed a molecular mass of 23,057.54 Da and when alkylated and reduced, the mass is 23,830.40 Da. Their peptides analyzed in MS (MALDI TOF\\TOF) showed significant score when compared with those of other proteins by NCBI-BLAST2 alignment display. As regards their proteolytic activities, BleucMP efficiently acted on fibrinogen, fibrin, and was inhibited by EDTA and 1.10-phenanthroline. This enzyme was also able to decrease significantly the plasma fibrinogen level provoking blood incoagulability, however was devoid of hemorrhagic activity when tested in the mice skin and did not induce relevant biochemical, hematological and histopathological alterations in mice. The aspects addressed in this paper provide data on the effect of BleucMP in envenomation from B. leucurus snakes in order to better understand the effects caused by snake venom metalloproteinase.