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Search for Plasma Proteins from Crotalus durissus terrificus snake responsible for protection against his own accidental envenomation and study of its potential use as antivenom

Cioce VM, Silva CS, Morais-Zani K, Sant´Anna SS, Grego KF, Tanaka-Azevedo AM

Laboratório de Herpetologia, Instituto Butantan, Brasil

Introduction: The toxicity of snake venoms is due to protein compounds with toxic action, such as myotoxic, neurotoxic and coagulant activities. Concerning the Crotalus snake venom composition, it presents crotoxin, crotamine, convulxin and gyroxin. It is known that the majority of snakes as well as some homeothermic animals have resistance against the venom of these venomous snakes and can survive in case of accidental envenomation.  Objectives: This present study investigates the plasma proteins from Crotalus durissus terrificus (C.d.t.) responsible for resistance against accidental envenomation by methods of purification and protein identification. Methods: The purification of the inhibitor from the serum of C.d.t. was performed by two chromatographic steps. As the first chromatographic step, an ion-exchange chromatography (DEAE-HiTrap – G.E.) was employed using C.d.t. serum, followed by affinity chromatography (Crotoxin coupled to Sepharose 4B – G.E.) with the preceding step pool fraction. The resulting fractions were analyzed by SDS-PAGE 1D (15%) and Western Blotting (W.B.) were performed using the isolated plasma inhibitor against venom and crotoxin. In addition, the purified protein was analyzed by mass spectrometry.  Results and Discussion: SDS-PAGE showed that the protein eluted from the affinity chromatography has a molecular mass around 20-25 kDa and 15-10 kDa (in reducing and no reducing condition, respectively). This work showed interaction of this inhibitor with the venom proteins and crotoxin by W.B., indicating that it may be responsible for the inhibition of venom toxins activity. After SDS-PAGE bands were extracted and digested by trypsin, they were identified by mass spectrophotometry as CNF, a phospholipase inhibitor present in the serum of the species C.d.t.

Supported by CNPq, FAPESP

10 - BIODIVERSITY 21th Annual Scientific Meeting of Instituto Butantan.