Figure 1.
Characterization of different SVMPs Bothrops neuwiedi venom.
Samples (150 µg) of B. neuwiedi venom were analyzed by 2-D gel electrophoresis in the pH range 3–10 and stained by Coomassie blue (A) or electrotransferred to nitrocellulose membranes and revealed with anti-jararhagin antibody followed by peroxidase labeled anti-rabbit IgG and enzyme substrate (B).
Figure 2.
Isolation of Bothrops neuwiedi venom fractions enriched in SVMPs.
(A) Samples of 50 mg of B. neuwiedi venom were applied on Hiprep 16/60 S200 column, equilibrated with 20 mM Tris, 150 mM NaCl and 1 mM CaCl2 pH 7.8. Fractions of 2.0 mL were collected at a flow rate of 0.5 mL/min, monitored at A280 (nm). (B,C and D) Fractions I, II and IV of size exclusion chromatography were applied to a Mono-Q HR 5/5, equilibrated with 20 mM Tris pH 7.8 plus 1 mM CaCl2. Fractions of 1.0 mL were collected at a flow rate of 1.0 mL/min, monitored at A280 (nm).
Figure 3.
SDS-PAGE and Western blotting of the isolated fractions.
Isolated fractions enriched with SVMPs (10 µg) were subjected to SDS-PAGE under non-reducing conditions and stained by Coomassie blue (A), silver stained (B) or electrotransferred to nitrocellulose membranes and revealed with anti-jararhagin antibody followed by peroxidase labeled anti-rabbit IgG and enzyme substrate (C). The bands with asterisks were cut and analyzed by mass spectrometry.
Table 1.
Assignment of the proteins isolated from the SDS-PAGE bands of B. neuwiedi venom fractions to protein families by MS/MS and MASCOT.
Figure 4.
Fibrinolytic, Gelatinolytic, Hemorrhagic activity and Recalcification time of SVMPs.
(A) Samples of 10 µg of each fraction was applied to the fibrin-agarose plate and the halos of lysis were measured. (B) Gelatinolytic activity of SVMPs (5 µg) on a 12.5% polyacrylamide gel copolymerized with gelatin. (C) Samples of 10 µg of each fraction were injected (i.d.) on the dorsum of mice followed by measuring of the hemorrhagic halo. (D) Samples with 0.1; 0.5; 1; 5 and 10 µg/mL SVMPs were incubated with platelet poor plasma. The reaction was initiated by addition of 0.025 M CaCl2 and the time to clot formation was measured.
Figure 5.
Prothrombin and Factor X activation by SVMPs fractions, as detected by S2238 and S2765 hydrolysis, respectively.
Samples (1 µg/mL) of isolated SVMPs were incubated with 2 µM prothrombin (A) or 0.4 µM FX (B). Thrombin and FXa formed was determined after 1, 5, 10, 15 and 20 minutes of reaction. Assay conditions and quantification of thrombin and FXa are described in the Material and methods. Each point represents mean ±SD of three determinations.
Figure 6.
ROTEM profile of citrated rat or chicken whole blood samples treated with SVMPs fractions.
Samples (40 µL) containing 10 µg SVMPs fractions were mixed with citrated rat or chicken whole blood samples (300 µL). ROTEM data are representative of three independent experiments and were acquired for 1 hour.
Table 2.
Relative comparison of functional activities of isolated fractions.