ISOLATION AND CHARACTERIZATION OF A THROMBIN-LIKE ENZYME FORM THE VENOM OF THE SNAKE BOTHROPS INSULARIS (JARARACA ILHOA)

Wermelinger, L.S.; Oliveira-Carvalho, A.L.; Castro, H.C.; Zingali, R.B.

Departamento de Bioquímica Médica, ICB, UFRJ

Objectives: B. insularis is a peculiar arboreal Brazilian snake found exclusively on Queimada Grande Island (São Paulo) that feeds only on birds. This venom presents a prominent fibrinoclotting activity (Selistre and Giglio, 1987). Our laboratory showed that ecotin, a trypsin/chymotrypsin inhibitor from E. coli, is able to interfere with this activity. Our main objective was to isolate and characterize the clotting factor from this venom that is affected by ecotin.

Methods and results: B. insularis crude venom was gel filtrated on Sephacryl S200HR column. The fraction that had fibrinoclotting activity affected by ecotin were further purified using an ecotin-agarose column. The eluted material (0.01 N HCl, 0.5 M NaCl, pH=2.0) showed a single band of activity in gelatin gel and also presented fibrinoclotting activity. Ecotin was able to enhance the fibrinoclotting activity by 50% with an EC₅₀= 91 microM.

Conclusion and perspectives: Here in, we describe the purification a thrombin–like enzyme from the venom of a B. insularis, interestingly this protein has its activity enhanced by ecotin. The sequence of this protein will be determined, the characterization and the mechanism of interaction with ecotin are now in progress.

Financial support: CNPq, FAPERJ.

CORRESPONDENCE TO:

Luciana Wermelinger Serrão, Rua Senador Vergueiro 250A, Apto. 906, Rio de Janeiro, RJ, Brasil, Email: luws@bol.com.br