Figure 1. Isolation of myotoxic phospholipases A₂ from the venom of Bothrops asper (Pacific versant of Costa Rica).

(A) Venom (200 mg) was applied to a CM-Sephadex C-25 column (20 × 2 cm) equilibrated with 0.05 M Tris, 0.1 M KCl, pH 7.0 and, after elution of unbound proteins, a linear gradient (G; dotted line) toward 0.05 M Tris, 0.75 M KCl, pH 7.0 buffer was developed. Proteins from peak 2 (horizontal green line) were pooled and further separated by (B) reverse-phase HPLC on a C₈ semipreparative column (250 × 10 mm). Proteins were eluted at 2.5 mL/min with a gradient from water to acetonitrile (dotted line) in the presence of 0.1% trifluoroacetic acid. The second major peak (∼24 min), corresponding to Asp49 phospholipases A₂, was collected and subjected to further analyses.