PARTIAL PURIFICATION OF METALLOPROTEASE FROM Bothrops jararacussu SNAKE VENOM

Mazzi, M.V., Esmeraldino, L.E., Buckeridge, Y.M., Veronese, E.L.G., Costa, S.M.C., Cintra, A.C.O., Sampaio, S.V.

Department of Clinical, Toxicological and Bromatological Analyses, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.

Objective: Isolation of metalloprotease for further biochemical and pharmacological characterization.

Methods and results: Crude B. jararacussu venom (CV) was fractionated on a Sephadex G-75 column, equilibrated and eluted with 0,05 M, pH 8,0 ammonium bicarbonate buffer at 25°C. The hemorrhagic fraction was rechromatographed on a DEAE-Sepharose column equilibrated and initially eluted with a 0,01 M, pH 7,2 phosphate buffer, followed by a NaCl concentration gradient which varied from 0.0 to 0.3 M in the same buffer. Hemorrhagic and clotting activities of the resulting subfractions were assayed according to Theakston and Reid, 1983. For the hemorrhagic assay the fractions were injected i.d. in 18-22g Swiss mice and the halos analyzed by the computer program “Image Tool”. For clotting assays, the fractions (25 mL) were added to 200mL of plasma and the time for first signs of fibrin formation recorded.  From gel filtration four fractions (SI to SIV) were obtained and only SI was hemorrhagic. Rechromatography of SI yielded seven subfractions (SIDI to SIDVII) among which SIDV, SIDVI and SIDVII were hemorrhagic, while SIDIV and SIDV were coagulant.

Conclusion: SIDV revealed the highest hemorrhagic activity, which was lost at low pH (3,5).

Financial support: CNPq and FAPESP.

CORRESPONDENCE TO:

Esmeraldino, L.E., Department of Clinical, Toxicological and Bromatological Analyses, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo. Avenida do café s/n. 14040-903, Ribeirão Preto, SP, Brazil, Email: luisee@usp.br