Isolation and Biochemical Characterization of a New Thrombin-Like Serine Protease from Bothrops pirajai Snake Venom
Figure 1
Purification profile of the serineprotease BpirSP-39 from Bothrops pirajai crude venom. The detached arrow (a) indicates the fraction with the highest coagulation activity, fraction 1 of 12.5% SDS-PAGE in denaturing conditions. Line 1: molecular mass standard, Color Plus Prestained Protein Marker, Broad Range (7–175 kDa) (P7709S New England Biolabs), lines 2–6: Fractions 1–5 obtained after chromatography. (b) Affinity chromatography of fraction 1 on benzamidine sepharose column. (c) High performance liquid chromatography using the C2/C18 column (10 mm × 4.6 mm, 3 m, 120 Å) and 12.5% SDS-PAGE of BpirSP-39 and B. pirajai crude venom. Lines 1 and 4: molecular mass standard: Protein Ladder (10–250 kDa) (P7703S New England Biolabs); 2- BpirSP-39 in denaturing conditions showing a band of approximately 49 kDa; 3-crude venom of B. pirajai in denaturing conditions. (d) Mass spectrum of BpirSP-39 determined by AXIMA TOF2. The identified protein presented a molecular mass of 39,408.32 Da. The peak at 19,579.02 Da indicates the double charge of the protein. The absorbance was monitored at A280 nm.
