Rapid Purification of a New P-I Class Metalloproteinase from Bothrops moojeni Venom with Antiplatelet Activity
Figure 1
Purification of the BmooMPα-II from B. moojeni venom. (a) Separation on DEAE-Sephacel ion-exchange chromatography: crude venom (400 mg) was applied on the column ( cm) and elution was carried out at a flow rate of 20 mL/h with ammonium bicarbonate gradients buffer (50 mmol/L–0.6 mol/L). Fractions of 3.0 mL/tube were collected and their absorbances at 280 nm were read. (b) Separation on benzamidine sepharose affinity chromatography: fraction DS7 was applied to the column previously equilibrated with 50 mmol/L Tris-HCl, 500 mmol/L NaCl, pH 7.4. After elution of the unbound fraction, 50 mmol/L glycine buffer, pH 3.0, was applied to the column and the absorbance of the fractions was monitored at 280 nm. Fractions of 3.0 mL/tube were collected at a flow rate of 30 mL/h. Pooled fractions are indicated by the closed circle. (c) SDS-PAGE in 14% (w/v) gel. Lanes: 1-standard proteins; 2-reduced BmooMPα-II; 3-non-reduced BmooMPα-II. The gel was stained with Coomassie blue R-250.
