Abstract
BackgroundBothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults.ResultsOptimized conditions for two-dimensional gel electrophoresis (2-DE) of pooled venom samples were achieved using immobilized pH gradient (IPG) gels of non-linear 3–10 pH range during the isoelectric focusing step and 10–20% gradient polyacrylamide gels in the second dimension. Software-assisted analysis of the 2-DE gels images demonstrated differences in the number and intensity of spots in juvenile, sub-adult and adult venoms. Although peptide mass fingerprinting (PMF) failed to identify even a minor fraction of spots, it allowed us to group spots that displayed similar peptide maps. The spots were subjected to a combination of tandem mass spectrometry and Mascot and MS BLAST database searches that identified several classes of proteins, including metalloproteinases, serine proteinases, lectins, phospholipases A2, L-amino oxidases, nerve growth factors, vascular endothelial growth factors and cysteine-rich secretory proteins.ConclusionThe analysis of B. atrox samples from specimens of different ages by 2-DE and mass spectrometry suggested that venom proteome alters upon ontogenetic development. We identified stage specific and differentially expressed polypeptides that may be responsible for the activities of the venom in each developmental stage. The results provide insight into the molecular basis of the relation between symptomatology of snakebite accidents in humans and the venom composition. Our findings underscore the importance of the use of venoms from individual specimen at various stages of maturation for the production of antivenoms.
Highlights
Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region
The analysis of B. atrox samples from specimens of different ages by two-dimensional gel electrophoresis (2-DE) and mass spectrometry suggested that venom proteome alters upon ontogenetic development
We identified stage specific and differentially expressed polypeptides that may be responsible for the activities of the venom in each developmental stage
Summary
Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. We present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults. Bothrops atrox species are responsible for the majority of snakebite accidents in the Brazilian Amazon region [2]. A comparative study of the proteinase activity and protein profiles of venoms from juvenile, sub-adult and adult Bothrops atrox specimens captured in the Brazilian Amazon rain forest was previously reported [2]. López-Lozano et al demonstrated that venoms from juveniles and sub-adults displayed higher human plasma clotting activity compared to adult venoms. Two proteins of 23 kDa and 50 kDa, respectively, that were present in higher amounts in adult venoms, were identified as metalloproteinases
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