Title: Isolamento, caracterização bioquímica e avaliação do potencial inflamatório de uma proteína secretada rica em cisteína (CRISP) da peçonha de Bothrops jararaca
Abstract: Envenomation by snakes from Bothrops genus is characterized by systemic and local effects such as coagulopathies, bleeding disorders, inflammation, pain and myonecrosis.The cysteine rich secretory proteins (CRISPs) are present in snake venoms and are widely distributed mammals, reptiles and amphibians.They are involved in certain biological activities, however many of their functions are still unknown.The aim of the present study was to isolate a CRISP from Bothrops jararaca and to biochemically/functionally characterize it by evaluating its involvement on inflammatory responses and on the complement system.The CRISP named BJ-CRP was isolated from Bothrops jararaca crude venom through the combination of three chromatographic steps: molecular exclusion on Sephacryl S-200 column, anion exchange chromatography on Source 15Q and reverse phase chromatography using C18 column.A high purity degree was obtained as confirmed by SDS-PAGE, showing a single band of 25.19 kDa, and by MALDI-TOF/MS showing a molecular mass of 24.6 kDa.The N-terminal sequence and analysis of tryptic peptides by MALDI TOF/ MS resulted in the determination of 100 amino acid residues, which had up to 96% similarity to sequences from other snake venom CRISPs that were previously described, but from other genus and snake species.The BJ-CRP did not have proteolytic activity on azocasein, fibrinogen or fibrin.It did not show coagulant or hemorrhagic activity, and also did not show activity on 13 different voltage dependent potassium channels when tested at a concentration of 1µM.Moreover, this toxin was able to induce an acute inflammatory response (1 and 4 hours after injection), observed by the recruitment of neutrophils and increase of interleukin-6 into the peritoneal cavity of mice.BJ-CRP and B. jararaca crude venom were capable of modulating the hemolytic activity promoted by the classical pathway of the complement system, and BJ-CRP also showed direct action on some complement system components, such as C3 and C4 as evaluated by SDS-PAGE and Western blot.The present work describes the purification of BJ-CRP, the first CRISP isolated from a Bothrops snake venom.The results obtained showed to be promising and open up prospects in order to better understand the involvement of this class of toxins in the inflammatory response induced by Bothrops envenomation.