Proteolytic activity of Bitis ssp venoms.[A] Gelatinolytic activity analyzed by zymography: Bitis spp venoms (30 μg) were incubated in the absence (buffer) or presence of 5 mM EDTA (EDTA), 5 mM 1,10-phenanthroline (PHE) or 5 mM PMSF (PMSF), submitted to electrophoresis and subsequently incubated overnight at 37°C, in 50 mM Tris-HCl, pH 8.3. Following incubation, the gel was stained with 0.1% Coomassie Brilliant Blue and the gelatinolytic activity was detected as unstained bands on a dark background. [B] Fibrinogenolytic activity was assessed using a SDS-PAGE (10%) gel. Thirty micrograms of human purified fibrinogen (Fn) were incubated with 1 μg of Bitis spp venoms pre-incubated or not with inhibitors of serine–(PMSF) or metallo–(PHE) proteinases and ran under reducing condition. The digestion of gelatin and the cleavage of fibrinogen were visualized by Coomassie Brilliant Blue staining.
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