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

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

ELISA reactivity with <i>Bothrops</i> antivenom and MAJar-3 monoclonal antibody of fractions collected from chromatograms of venom from snakes classified in different genera.

<p>Samples containing 100 µL 1-µg/mL fractions collected at the elution times represented in the chromatograms were used to coat maxisorb microplates (Nunc), which were incubated with SAB (1∶1,000) or a monoclonal antibody against jararhagin (class P-III SVMP) MAJar-3 (1∶50), followed by incubation with anti-horse IgG (1∶2,000) or anti-mouse IgG (1∶1,000) labeled with peroxidase. The reactions were developed with ortho-phenylenediamine/H₂O₂ as the enzyme substrate, and the products were detected at 490 nm. The ELISA reactivity was calculated as % reactivity, taking as 100% the maximal OD value obtained in each of three independent experiments performed in duplicate.</p

Protein family distribution for the venoms of the three different snake genera, as determined using a shotgun proteomics approach.

<p>Each venom sample was prepared in duplicate, and the MS analyses were performed in triplicate for each venom sample replicate (a total of six MS analyses per venom). The data represent the mean of the normalized total spectral count distributed as follows: 1,891 (<i>B. atrox</i>); 1,727 (<i>B. jararacussu</i>); 2,719 (<i>B. jararaca</i>); 2,287 (<i>B. neuwiedi</i>); 1,252 (<i>R. alternatus</i>) and 1,767 (<i>R. cotiara</i>). The following were identified: SVMP-I, SVMP-II and SVMP–III (snake venom metalloproteinase - classes P-I, P-II and P-III); PLA2 (phospholipase A2); SVSP (snake venom serine proteinase); CLEC (C-type lectin); CLECL (C-type lectin-like); LAAO (L-amino acid oxidase); NGF (nerve growth factor); HYALU (hyaluronidase); VEGF (vascular endothelial growth factor); CRISP (cysteine-rich secretory protein); PDIEST (phosphodiesterase 1); ECTONT (ecto-5′-nucleotidase); PLB (phospholipase B); GLUTCYC (glutaminyl cyclase) and ACTIN (actin).</p

Comparison of ELISA titration curves of <i>Bothrops</i> antivenom with venom from snakes classified in different genera.

<p>Samples containing 100 µL whole venom (10 µg/mL) were used to coat maxisorb microplates (Nunc), which were incubated with crescent dilutions of SAB (starting from 1∶10,000), followed by incubation with anti-horse IgG labeled with peroxidase (1∶2,000). The reactions were developed with ortho-phenylenediamine/H₂O₂ as the enzyme substrate, and the products were detected at 490 nm. The experiments were performed in duplicate in three independent experiments, and the results are expressed as the mean ± sd of the six OD values.</p

Neutralizing ability of <i>Bothrops</i> antivenom (SAB) against the major toxic activities of <i>Bothropoides jararaca</i> and <i>Bothrops atrox</i> venoms.

<p>In the neutralization assays, the <i>Bothrops atrox</i> venom was pooled from 8 adult snakes collected in FLONA Tapajós, Santarém, Pará, Brazil. For the neutralization of lethality and hemorrhagic activity, doses of <i>B. jararaca</i> or <i>B. atrox</i> venoms were pre-incubated with SAB at ratios of 1, 2 or 4 times the SAB volume required to neutralize an equal amount of reference venom according to the manufacturer. To assess hemorrhage, 10 µg was incubated and injected intradermically in the dorsum of a group of 5 mice. The results show the % neutralization of the mean values, taken as 100% activity, of the data obtained after injection with venom incubated with saline. For the neutralization of lethal activity, 3 LD₅₀ doses of <i>B. jararaca</i> (105 µg) or <i>B. atrox</i> (225 µg) venom were incubated, and the mixtures were injected intraperitoneally into groups of 5 mice; lethality was recorded over a period of 48 hours. The results represent the values obtained in 3 independent experiments and are expressed as % neutralization, considering the number of live/dead mice after this period. To assess the neutralization of coagulant activity, a constant amount of venom (2 times the minimum coagulant concentrations) was incubated with several dilutions of antivenom; the mixture was added to 100 µl bovine plasma, and the clotting times were recorded using a model ST4 mechanical coagulometer (Diagnostica Stago). The neutralization was expressed as the effective dose (ED), defined as the antivenom/venom ratio at which the clotting time was increased threefold when compared to the clotting time of plasma incubated with venom alone.</p