Abstract
Serineproteases and metalloproteases are the main Bothrops jararaca venom enzymes acting on the victim’s tissues and proteins. As a result of their direct actions on tissue proteins, these proteases could generate peptides with specific actions in cells or other mechanisms. The most common sources for bioactive peptides are natural precursor proteins. Recent studies have shown that a new class of proteins not named as precursors, the crypteins, may, in some conditions, originate bioactive peptides, or cryptides. New cryptides generated by the action of the venom serinoproteases and by commercial trypsin on endogenous substrates, were isolated, then biochemically and biologically characterized. Serineproteases from B. jararaca venom were separated from the whole venom using an HPLC molecular exclusion column, verifying the activity of the fractions on the chosen substrates (myoglobin, hemoglobin, immunoglobin G and collagen). These substrates were incubated with the venom serineproteases as well as with trypsin. The resulting peptides were separated by fractionation by HPLC and the fractions were tested on cell cultures for proliferative or cytotoxic effects. Active fractions were rechromatographed in order to obtain the pure bioactive peptides. After the activity was confirmed, the peptides were sequenced and synthesized. Trypsin activity on myoglobin generated peptides (ALELFR, TGHPETLEK, GLSDGEWQQVLNVWGK) presenting proliferative activity on fibroblasts and endothelial cells. 3D modeling of myoglobin, using Cn3D software, showed that the three peptides are located on the surface of the protein. Bioactive fractions were also found after digestion of the other substrates mentioned above with trypsin, but they were not yet isolated and sequenced. Digestion of myoglobin with the venom serineproteases generated an HPLC profile similar to the one obtained with trypsin. This suggests that the cryptides here described may indeed be generated at the the snake bite site, causing secondary effects, not neutralized by serumtherapy. This study suggests that the venom serineproteases may generate cryptides with relevant effects through their actions on highly available protein substrates at the bite site.
This work is licensed under a Creative Commons Attribution 4.0 International License.
Copyright (c) 2013 Aline Vivian Vatti Auada, Ivo Lebrun (Orientador)