Isolation and enzymatic studies of a new thrombin-like enzyme BaIII-4 isolated from Peruvian Bothops atrox venom 

Ponce-Soto, L.A.(1), Toyama, M.H.(1,2), Novello, J.C.(1), Yarlequé Chocas, A.(3), Marangoni, S.(1)

(1)Departaments of Biochemistry, (2)Physiology and Biophisics Institute of Biology State University of Campinas (UNICAMP) Campinas, SP, Brazil, (3)Faculty of Biological Sciences, National University of San Marcos (UNMSM), Lima, Perú.

Objective: A new thrombin-like serine protease was isolated from the venom of Bothrops atrox and was partially characterized.

Methods and Results: The thrombin-like enzyme was isolated from B. atrox venom using molecular exclusion and reverse phase HPLC and mol mass of ~34 kDa in Tricine SDS-PAGE 12.5%.

The enzyme purified BaIII-4 had a high content of Asx, Glx, Gly, Ser, Ala and Pro, with 12 half Cys. The N-terminal amino acid sequence was: VIGGD ECDIN EHPFL AFMYY SPRYF CGMTL INQEW. BaIII 4 had a Km and Vmax of K_m 7.1 X 10^{-4  M} and V_max3.8 X 10^-2 nmolesp-NA/min/mg. The enzymatic and biological activities (fibrinogenolitc action and platellet aggregation) of the enzyme were inhibited by protein aprotinin, PMSF, soybean trypsin inhibitor, TLCK and DA2 II (on anti-hemorrhagic protein from Didelphis albiventris opossum serum).

Conclusion: BaIII-4 showed similar Km and Vmax to those of thrombin-like enzymes from snake venoms. The inhibition by PMSF, TLCK, aprotinin and soybean trypsin inhibitor suggest a trypsin-like catalitic mechanism.

Financial supported by: FAPESP

CORRESPONDENCE TO:

Luis Alberto Ponce-Soto, Avenida Santa Isabel, 1125 Bloco B-2, Campinas, SP, CEP: 13084-970, Brasil, Email: pelao01@hotmail.com