Poster 155 - Ion exchange chromatography of Bothrops moojeni venom and some biochemical proprieties

Poster 155. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.514.

ION EXCHANGE CHROMATOGRAPHY OF Bothrops moojeni VENOM AND SOME BIOCHEMICAL PROPRIETIES

¹Manfiolli, A. O.; ¹Vieira, L. F.; ¹Menezes, C.; ²Costa, J. de O.; ¹Oliveira, F.

1 Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Minas Gerais, Brasil. 2 Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, Minas Gerais, Brasil.

In this work we describe the purification and some biochemical proprieties of the Bothrops moojeni venom. Crude venom of B. moojeni (200mg) was chromatographed on DEAE Sephacel column, previously equilibrated with 0.05M, pH7.8 ammonium bicarbonate. The chromatography resulted in six fractions denominated D1-D6. Protein concentrations were determined by the method of Microbiuret. Clotting activity was determined by mixing 10mg of each fraction (D1-D6) with 200mL of plasma and fibrinogen bovine, at 37ºC, and determining the clotting time. The hydrolytic activity of the fractions on fibrinogen was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after incubating in 0.150M Tris–HCl, pH8.0, buffer with 0.001g of fractions and 0.01g of fibrinogen for 60min at 37°C. Defibrinating activity was evaluated after administering i.p. 50mg of each fraction in Swiss mice. After one hour, animals were anesthetized with ether and bled by cardiac puncture. Whole blood was placed in tubes and kept at 25–30°C until clotting occurred. Proteolytic activity was tested on azocasein as substrate (1mg/ml 0.2M Tris/HCl, pH8.8, 0.004M CaCl2). A_{366 nm} is a measure of the degree of proteolytic degradation of azocasein by the sample. The crude venom and the fractions D3 and D4 showed blood-clotting activity on plasma and fibrinogen bovine, while the other fractions had not presented this activity. Crude venom and all fractions showed a high proteolytic activity against A-a, B-b chains of bovine fibrinogen. The crude venom and the fractions D1, D2, D3 and D4 acted on azocasein with specific activity of 1,530, 820, 980, 750 and 710 units/mg, respectively. In addition, the crude venom, D1, D2, D3 and D4 caused defibrinogenation when administered i. p. to mice, making the plasma uncoagulable. Our results showed that the crude venom from the Bothrops moojeni is rich in proteolytic enzymes.