Isolation of a new toxic protein from the Leptodactyluspentadactylus(Amphibia, Anura) skin secretion

EVANGELISTA, J. S. M., HONÓRIO-JÚNIOR, J.E.R., FALCÃO, N.R., OLIVEIRA, R.G.S., CARDI; B.A., CARVALHO, K.M.

Laboratório de Neurofarmacologia, Universidade Estadual do Ceará, Fortaleza, CE, Brazil.

Objectives: Amphibians have undergone profound evolutionary changes to survive to predators and microorganisms, and their skin secretions containing toxins, alkaloids, biogenic amines, proteins, peptides, seems to be a paramount importance. In this paper, we demonstrated the purification and pharmacological effects of a new toxic protein from L. pentadactylus skin secretion.

Methods and Results: Skin secretion was obtained after injection of 500ml adrenaline (100mg/ml, s.c.) in dorsal skin. After dilution in bidestilated water (1:4, v:v) and lyophilization, it was kept at -25°C. The toxin was purified by Sephacryl S-400 chromatography and its purity was evaluated by denaturing and non-denaturing gel electrophoresis. The LD₅₀, pharmacological and toxicological analysis were evaluated after i.v. injection on mice. On SDS-PAGE only 29 kDa form was identified. The LD_{50 values were 1.0}±0.2 mg/Kg (crude venom) and 0.5±0.03 mg/Kg (purified toxin), the dead time was less than 1 minute. The preliminary histopathological analysis showed pulmonary congestion macroscopically, without significant microscopical changes at this place or on other organs. In vitro hemolysis (washed mouse erythrocytes), using crescent toxin concentrations (0.012-0.12mg/ml), was observed. At 0.5-1.0 mg/Kg doses rate, we observed locomotion activity decreased, sub-convulsion movements, prostration, defecation/urination increased, nasal discharge, mydriasis, and dyspnea. At low doses (< 0.5 mg/Kg), the toxin induced in vivo an acute and reversible decrease of the blood pressure and in vitro an inotropic positive effect on heart, without interfering on the frequency.

Conclusions:  Although the mechanism of action of this toxin is unknown, these results suggest that it may be a tool to study the cardiovascular functions involved on blood pressure and heart homeostasis.

CORRESPONDENCE TO:

Bruno Andrade Cardi, Rua Monsenhor Dantas, 1931/402, Fortaleza, CE, CEP: 60310-220, Brasil, Email: bcardi@bol.com.br