Laboratório de Toxinologia

In this work, we have developed a simpler and faster methodology to isolate two metalloproteinases from Bothrops jararaca venom, presumably jararhagin (52kDa) and bothrolysin (23kDa). B. jararaca venom (10mg) was submitted to molecular exclusion chromatography on Superdex 200 HR 10/30 and the chromatographic profile displayed three major fractions at 280nm. As expected, the first fraction contained protein bands with higher molecular masses, including a band compatible with the molecular mass of jararhagin. This fraction was further submitted to anion exchange chromatography on a Mono-Q HR 5/5 column with a linear salt gradient up to 0.5 M NaCl. The Mono-Q chromatography resulted in homogeneous jararhagin obtention, visualized by SDS-PAGE 12,5%. Furthermore, the third fraction obtained by molecular exclusion was also submitted to ion-exchange chromatography (as described above). SDS-PAGE 12.5% revealed a homogeneous low molecular weight component compatible with the bothrolysin molecular mass. All purification steps were performed at 4°C. These two homogeneous proteins displayed proteolytic activity over gelatin, visualized by zimography, revealing their enzymatic nature. These results have demonstrated a new purification methodology to acquire these unstable enzymes in a simpler and faster manner. These isolated enzymes will be used as tools in several projects in our laboratory.