Chromatography of stingray PLA2 on FPLC Mono-S Sepharose and Mono-Q Sepharose. (A) Chromatography of stingray PLA2 on FPLC Mono-S Sepharose. The column (2.6 cm × 20 cm) was equilibrated with 100 mM acetate buffer, pH 4.5, containing 0.05% Triton X-100 and 2 mM benzamidine (buffer A); a linear salt gradient (0.1 to 0.4 M NaCl) in buffer A was applied to the column; gradient chamber 100 ml; 2 ml fraction; flow rate, 30 ml/h. (B) Chromatography of stingray PLA2 on Mono-Q Sepharose step. The column (1.5 cm × 20 cm) was equilibrated with 25 mM tris-HCl buffer, pH 8, containing 25 mM NaCl. Proteins were eluted by three washes applied to the column (3 × 80 ml from 100 mM to 300 mM NaCl). The flow rate was 40 ml/h and the fraction size was 4 ml. SPLA2 activity was measured as described in materials and methods using PC as substrate. Active fractions (28 to 34) were gathered.