ISOLATION AND PARTIAL CHARACTERIZATION OF A HEMORRHAGIC PROTEIN FROM Bothrops lanceolatus VENOM

Stroka, A., Donato, J. L., Hyslop, S., Lôbo de Araújo, A.

Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas, UNICAMP, Campinas, SP. 

Introduction: Bothrops lanceolatus venom contains hemorrhagic metalloproteinases that can be inhibited by EDTA. In this work, we have purified one of these proteins. Methods and Results: Fraction FI obtained by gel filtration was chromatographed on Q-Sepharose preequilibrated with 0.05 M Tris-HCl, pH 7.5. The proteins were eluted (30 ml/h) with a linear gradient (0-1.0 M) of NaCl in this buffer. The active fraction (FI2) with caseinolytic and hemorrhagic activities was dialyzed  against Tris-HCl, pH 7.5, and applied to a column of Blue-Sepharose that was then eluted as described above. In all cases, the elution profile of the proteins was monitored at 280 nm. Fractions FI2, FI2a and FI2b were analyzed by SDS-PAGE in 12% gels. Fractionation of FI on Q-Sepharose resulted in a major peak (FI2) which contained all of the caseinolytic and hemorrhagic activities. Chromatography of this peak on Blue-Sepharose resulted in two fractions (FI2a and FI2b), and both had  caseinolytic and hemorrhagic activities. However, only FI2b showed a single band following SDS-PAGE. The estimated molecular mass of this protein was 53.3 kDa. Conclusion: Bothrops lanceolatus venom apparently contains more than one hemorrhagic protease, as shown  by two peaks obtained following affinity chromatography on Blue-Sepharose. The co-elution of caseinolytic and hemorrhagic activities and the molecular mass of the protein in fraction FI2b agreed with observations for other Bothrops venoms.

Financial support: FAPESP and FAEP.

CORRESPONDENCE TO:

Lôbo de Araújo, A., Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), CP 6111, Campinas, SP, Email: albetiza@fcm.unicamp.br