Sequence alignment of the serine proteinases of the venom of <i>S</i>. <i>c</i>. <i>edwardsii</i>.

<p>Different colors indicate the unique peptides identified by tandem mass spectrometry of the corresponding proteins.</p

Identification of proteins of the venom of <i>S</i>. <i>c</i>. <i>edwardsii</i> not predicted from the transcriptome analysis.

<p>Identification of proteins of the venom of <i>S</i>. <i>c</i>. <i>edwardsii</i> not predicted from the transcriptome analysis.</p

Sequence alignment of the metalloproteinases of the venom of <i>S</i>. <i>c</i>. <i>edwardsii</i>.

<p>Different colors indicate the unique peptides identified by tandem mass spectrometry of the corresponding proteins.</p

Sequence coverages of some of the predicted venom gland proteins of <i>S</i>. <i>c</i>. <i>edwardsii</i> as revealed by the combined approach of MALDI and ESI tandem mass spectrometry.

<p>Protein sequences including specific domains are indicted by colored bars; below these, corresponding peptides identified by ESI (black lines) and MALDI (red lines) are indicated.</p

XA concentration increases in the midgut ABM owing to an increase in the kynurenine pathway.

<p>(A) The midguts of adult females were dissected after feeding the insects with rabbit blood (•) or with plasma (○) at the indicated times, and the XA content was evaluated using HPLC. (B) The insects were fed with blood plus 1 mg/ml 1-methyl-tryptophan, compound Ro-61-8048 or m-NBA, and the XA content was evaluated at 12 h and 24 h ABM. * indicates p<0.05 using Student's t-test. Data shown are mean ± SEM (n = 4).</p

XA is an abundant component of the <i>Aedes aegypti</i> midgut.

<p>(A) HPLC profile of a midgut extract from RED strain (WT) and WE strain insects 24 h ABM (one midgut was used in each run). The inset shows an HPLC run with standards of kynurenine (KYN), xanthurenic acid (XA), kynurenic acid (KYNA) and tryptophan (TRIP). (B) Light absorption spectra of the XA peak from the WT midgut (solid line) and of the kynurenic acid peak from the WE midgut (dotted line). (C) ESI-MS of the XA [M+H]⁺ peak collected from the midgut HPLC fractionation (shown in B) with m/z 206.1 revealed a molecular mass of 205 Da. (D) MS² of m/z 206.1 produced m/z 178.2 that could correspond to the loss of the formic acid plus a water addition. (E) MS³ of m/z 178.2 produced m/z 160.0 and 132.2 among others. (F) ESI-MS of the kynurenic acid peak collected from the WE midgut HPLC fractionation (shown in A) displaying m/z 190.050. (G) MS² of m/z 190.050 produced m/z 173.000, 162.055 and 144.045, which are identical to those formed from standard kynurenic acid (not shown). The MS³ of the m/z 173.000 (172.0397) did not provide additional species (not shown).</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

Comparison of electrophoretic profile (A) and <i>Bothrops</i> antivenom antigenic reactivity (B) of venoms from snakes classified in different genera.

<p>Samples containing 10 µg <i>Bothropoides jararaca</i> (JAR), <i>Bothropoides neuwiedi</i> (NEU), <i>Bothrops atrox</i> (ATR), <i>Bothrops jararacussu</i>(JSU), <i>Rhinocerophis alternatus</i> (ALT) and <i>Rhinocerophis cotiara</i> (COT) venoms were fractionated by SDS-PAGE (12.5% acrylamide gels) under non-reducing conditions and were either stained with Coomassie blue (<b>A</b>) or transferred to nitrocellulose membranes, which were then incubated with SAB (1∶1,000) as the primary antibody and peroxidase-labeled goat anti-horse IgG (1∶1,000). The reactive bands were detected by incubation with 4-chloro-α-naphthol and H₂O₂ (<b>B</b>). The numbers at the left indicate the mobility of the molecular mass markers in kDa. These results represent three independent runs.</p

Comparison of the elution profiles of venoms from snakes classified in different genera.

<p>Samples containing 5 mg of crude lyophilized venom from <i>Bothrops atrox</i>, <i>Bothrops jararacussu</i>, <i>Bothropoides jararaca</i>, <i>Bothropoides neuwiedi</i>, <i>Rhinocerophis alternatus</i> and <i>Rhinocerophis cotiara</i>, species maintained at Instituto Butantan herpetarium, were applied to a Vydac C-18 column (4.6×250 mm, 10-µm particle size) coupled to an Agilent 1100 HPLC system. The fractions were eluted at 1 mL/min, with a gradient of 0.1% TFA in water (solution A) and 0.1% TFA in acetonitrile (solution B) (5% B for 10 min, followed by 5–15% B over 20 min, 15–45% B over 120 min, 45–70% B over 20 min and 70–100% B over 10 min). The separations were monitored at 214 nm.</p