Purification and Characterization of BmooAi: A New Toxin from Bothrops moojeni Snake Venom That Inhibits Platelet Aggregation
Figure 1
Sequential purification steps of BmooAi from Bothrops moojeni venom. (a) Ion-exchange chromatography on a DEAE-Sephacel column: crude venom (400 mg) was applied to the column ( cm) and elution was carried out at a flow rate of 20 mL/h with ammonium bicarbonate gradient buffer (50 mmol/L–0.6 mol/L). Fractions of 3.0 mL/tube were collected and their absorbance read at 280 nm. (b) Molecular exclusion on a Sephadex G-75 column: the active fraction (DS4) was applied to the column and eluted with 50 mmol/L ammonium bicarbonate buffer at pH 7.8 with a flow rate of 20 mL/hour. (c) Reverse-phase HPLC chromatography on a 2.0 × 2.5 cm C2/C18 column (GE Health Care), equilibrated with solvent A (0.1% trifluoroacetic acid) and eluted with a concentration gradient of solvent B (80% acetonitrile and 0.1% trifluoroacetic acid) from 0 to 100% at a flow rate of 0.5 mL/min at room temperature. (d) SDS-PAGE in a 14% (w/v) gel. Lanes: 1: standard proteins; 2: reduced BmooAi fraction; 3: nonreduced BmooAi fraction. The gel was stained with Coomassie blue R-250.
