Partial isolation and characterization of thrombin-like BuII-3 from the venom of Bothropsalternatus

Ribeiro, D.A.(1), Toyama, M.H.(1,2), Ponce-Soto, L.A.(1), Marangoni. S.(1), Novello, J.C.(1)

(1)Departments of Biochemistry, (2)Physiology and Biophisics Institute of Biology State University of Campinas (UNICAMP) Campinas, SP Brazil.

Objective: In this work we showed the purification and partial characterization of thrombin-like isolated from the venom of Bothrops alternatus.

Methods and Results: This clotting serine protease (BuII-3) was isolated using a combination of molecular exclusion and reverse phase HPLC, respectively.

The molecular mass homogeneity was showed by tricine SDS-PAGE that in presence or absence of DTT reveled an electrophoretical band of approximately 31 kDa.

This protein showed high catalytic activity on the DL – BAPNA (N-Benzoyl-DL-arginine p-nitroanilide) as well as on Aa-chain of bovine fibrinogen, both assayed at 37oC in presence of Ca⁺². The N-terminal sequence of BuII-3 was IIPPVVINEHRSLVAI. Amino acid analysis showed Asx/42, Glx/29, Ser/21, Gly/18, His/8, Arg/12, Thr/10, Ala/22, Pro/17, Tyr/11, Val/17, Met/4. halfCys/5, Ile/18, Leu/21, Phe/21 and Lys/21. BuII-3 showed high content of Asx and Glx and five Cys, suggesting an acid character of this protein. This protein showed similar disulfide bridges pattern found in other thrombin-like.

Conclusion: In conclusion, the purification protocol described here provided an efficient recovery of this enzyme can yield sufficient amounts of enzyme for detailed studies of its biological activities. Bu-II-3 showed similar structural and fibrino(geno)litic serine protease to those of thrombin-like enzymes from snake venoms.

Financial Support: CAPES, CNPq and FAPESP

CORRESPONDENCE TO:

DulcinéiaAparecida Ribeiro, Avenida Santa Isabel, 1338, Campinas, SP, CEP: 13084471, Brasil, Email: dribeiro@unicamp.br