Purification of BmooPAi from B. moojeni snake venom. (a) Separation on DEAE-Sephacel ion-exchange chromatography: crude venom (200 mg) was applied to the column (2.5 × 20 cm) and elution was carried out at a flow rate of 20 mL/h with ammonium bicarbonate (Ambic) buffer gradients, pH 7.8, from 0.05 M to 0.6 M. Fractions of 3.0 mL/tube were collected and the absorbance was read at 280 nm. (b) Separation on Sephadex G-75 molecular exclusion chromatography: fraction D7 was applied to the column (1.0 × 100 cm) and elution with 0.05 M ammonium bicarbonate was achieved at a flow rate of 20 mL/h. Fractions of 3.0 mL/tube were collected and the absorbance was read at 280 nm. (c) Separation by affinity chromatography on a HiTrap Heparin HP column using the ÄKTApurifier HPLC system: fraction D7S2 was applied to the column (5 × 1 mL), previously equilibrated with 20 mM Tris-HCl buffer (pH 7.0) containing 5 mM calcium chloride. The samples were eluted with an increasing concentration gradient of 20 mM Tris-HCl buffer (pH 7.0) containing 2.0 M sodium chloride, and the absorbance of the fractions was monitored at 280 nm. Fractions of 1.0 mL/tube were collected at a flow rate of 30 mL/h. (d) SDS-PAGE in 14% (w/v) polyacrylamide, Tris-glycine buffer, pH 8.3, and 20 mA. Lanes: 1, standard proteins; 2, reduced crude venom of B. moojeni; 3, reduced BmooPAi; 4, nonreduced BmooPAi. The gel was stained with Coomassie blue R-250. (e) Reverse-phase HPLC on a C2C18 column (4.6 × 100 mm) equilibrated with 0.1% trifluoroacetic acid (TFA) and eluted with a linear concentration gradient from 0 to 100% of solution B (70% acetonitrile in 0.1% TFA).