Efeitos da autoproteólise e de fatores físico-químicos nas atividades biológicas e antigenicidade das toxinas do veneno da serpente amazônica Bothrops atrox (Linnaeus, 1758) “JARARACA”.

Abstract

In Brazilian Amazonian rain forest venomous snake Bothrops atrox (Viperidae) are responsible for 90% of snakebites. Its venom has anticoagulant, proteolytic and hemorrhagic (local and systemic) biological activities. Proteolysis and physicochemical factors (pH changes, temperature, composition of solvent) and the time of the agent's action can affect the three-dimensional structure/conformational state of a native protein, which could lead to the emergence of proteins and peptides with different biological activities and antigenic properties when comparational with original protein. Literature review found no information about these influences on the biological activities and antigenicity of toxins from venom of B. atrox venom. Because of this, this work provided to evaluate the biological activities and antigenicity of toxins from the venom after autoproteolysis at 37°C in different buffer solutions (PBS - 100 mM, pH 7.2 and COLLAGENASE, 50 mM Tris, pH 7.5) incubation time was until one week. The results showed that the molecular profiles, the biological activities and antigenicity of toxins from B. atrox autoproteolisysed second incubation time were strongly influenced by the constituents of the buffer solutions used in the experimental process. Brij 35, a constituent of the buffer COLLAGENASE, protected mainly from proteolytic degradation and freeze-thaw, the PI class metalloproteinase (23 kDa) and P-III (50 kDa) of the venom, preserving the structure / biological function of them, thus influencing the high hemorrhagic activity, toxicity and lethality of the venom. The venom of B. atrox autoproteolysed in PBS showed decay total amount of the toxin as seen in the molecular profiles, and rapid degradation of hemorrhagic metalloproteinase-class P-III, during the time of incubation at 37°C, causes higher decay hemorrhagic, proteolytic, edema and lethal activities. The experimental hyperimmune serum obtained by using as antigen a sample of B. atrox venom autoproteolysed for a week at 37°C in PBS showed satisfactory efficacy neutralizing activity of hemorrhagic, proteolytic and lethal activities from in natura B. atrox venom, and also low toxicity in the process of immunization of laboratory animals. The results suggest the purification of non-toxic peptides/proteins from B. atrox venom proteolysis at 37°C by their own proteases for a week in PBS buffer (100 mM), pH 7.2 evaluating its antigenic activity generating subsidies for vaccine production / specific antivenom against the venom of the Amazonian snake B. atrox.