Characterization of different SVMPs <i>Bothrops neuwiedi</i> venom.

<p>Samples (150 µg) of <i>B. neuwiedi</i> 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).</p

SDS-PAGE and Western blotting of the isolated fractions.

<p>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.</p

Prothrombin and Factor X activation by SVMPs fractions, as detected by S2238 and S2765 hydrolysis, respectively.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109651#s2" target="_blank">Material and methods</a>. Each point represents mean ±SD of three determinations.</p

Relative comparison of functional activities of isolated fractions.

<p>The biological activities shown above were classified as low (+), moderate (++), or high (+++) for graphical representation.</p><p>Relative comparison of functional activities of isolated fractions.</p

Assignment of the proteins isolated from the SDS-PAGE bands of B. neuwiedi venom fractions to protein families by MS/MS and MASCOT.

<p>X =  Leucine or Isoleucine, B =  Lysine or Glutamine,</p><p>*Found only in Bothropasin sequence,</p><p>**Found only in Jararhagin sequence. C- or N-terminal peptides are underlined.</p><p>Assignment of the proteins isolated from the SDS-PAGE bands of B. neuwiedi venom fractions to protein families by MS/MS and MASCOT.</p

Fibrinolytic, Gelatinolytic, Hemorrhagic activity and Recalcification time of SVMPs.

<p>(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.</p

Isolation of <i>Bothrops neuwiedi</i> venom fractions enriched in SVMPs.

<p>(A) Samples of 50 mg of <i>B. neuwiedi</i> 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).</p

ROTEM profile of citrated rat or chicken whole blood samples treated with SVMPs fractions.

<p>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.</p

Principal Component Analysis relative to toxin composition.

<p>Loading (top) and score (bottom) plots of the principal components 1 and 2 of the venoms from <i>Bothrops atrox</i> (ATR), <i>Bothrops jararacussu</i> (JSU), <i>Bothropoides jararaca</i> (JAR), <i>Bothropoides neuwiedi</i> (NEU), <i>Rhinocerophis alternatus</i> (ALT) and <i>Rhinocerophis cotiara</i> (COT) according to their protein composition including as variables the normalized maximal mAU at 214 nm in defined elution intervals of C-18 reverse-phase chromatography (Panel A), or the normalized total spectral counts of each protein group, as evaluated by shotgun mass spectrometry (Panel B). The Principal Component Analysis was based on the covariance matrix and all calculations were carried out in the software Minitab 16.</p

Venom clustering according to toxin composition.

<p>The venoms from <i>Bothrops atrox</i> (ATR), <i>Bothrops jararacussu</i> (JSU), <i>Bothropoides jararaca</i> (JAR), <i>Bothropoides neuwiedi</i> (NEU), <i>Rhinocerophis alternatus</i> (ALT) and <i>Rhinocerophis cotiara</i> (COT) were classified according to their protein composition by hierarchical clustering of the observations, including as a variable the normalized maximal mAU at 214 nm in defined elution intervals of C-18 reverse-phase chromatography (Panel A) or normalized total spectral counts of each protein group, as evaluated by shotgun mass spectrometry (Panel B). The procedure used an agglomerative hierarchical method linked by the minimum Euclidean distance between an item in one cluster and an item in another cluster (nearest neighbor) using the Minitab 16 software.</p