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Protein isolation from Bothrobs jararaca serum with capacity of venom inhibition.

Silva CS, Grego KF, Morais-Zani K, Tanaka-Azevedo AM

Laboratório de Herpetologia, Instituto Butantan, Brasil

Introduction: The phospholipase A2 (PLA2) are enzymes that act on cell membrane phospholipids carrying out the cleavage of fatty acids and lysophospholipids, deconstructing the cell wall. This protein is commonly found in snake venoms, causing tissue inflammation in the area affected by the sting. Evidence indicates that snakes have natural resistance due protective properties of plasma that inhibit the action of proteins present in the venom.  Objectives: This study aimed to purify and characterize proteins present in the plasma of the Bothrops jararaca snakes species with ability to inhibit PLA2 present in venom. Methods: For purification of the inhibitor, the crotoxin, abundant protein on the venom of Crotalus durissus terrificus, was coupled to a CNBr-activated Sepharose resin. The serum B. jararaca was applied to this column that had been equilibrated in 137mM NaCl 2.7mM KCl 9.1mM Na2HPO4 2H2O 1.8mM KH2PO4, pH 7.4 (PBS). Then the proteins with affinity to column were eluted with a gradient PBS 1M and Glycine pH 2.0. The purified protein by affinity chromatography was analyzed by mass spectrometry and evaluated for its ability to inhibit by adapting the method described by Holzer and Mackessy (1996). Results and Discussion:  In SDS-PAGE the protein eluted from the affinity chromatography showed a mass of 150kD without reducing buffer and 25kDA with reducing buffer. According to analysis by mass spectrometry it has 72% and 68% coverage of the amino acid sequence for the two proteins already described as phospholipase A2 inhibitors, and also was capable of inhibiting the phospholipase activity when incubated with B. jararaca venom.  CONCLUSION: It was purified a protein with phospholipase A2 inhibitors characteristics that may represent an important alternative for the neutralization process of poison.

Supported by Capes, CNPq, FAPESP



2 - BIOCHEMISTRY 21th Annual Scientific Meeting of Instituto Butantan.