PURIFICATION AND CHARACTERIZATION OF LOW-MOLECULAR-WEIGHT FIBRINOLYTIC HEMARRHAGIC ENZYMES FROM SNAKE (BOTHROPS-JARARACA) VENOM/

Two low molecular weight fibrinolytic/hemorrhagic enzymes, jararafibra se III and jararafibrase IV, were purified from Bothrops jararaca veno m using a fast protein liquid chromatography system. The purified jara rafibrase III and jararafibrase IV were single chain proteins with mol ecular weights of 20,400 +/- 500 and 21,200 +/- 400, respectively, by SDS-PAGE. The isoelectric points of jararafibrase III and jararafibras e IV were 9.4 and 6.9, respectively. The activity of the enzyme was in hibited by 1,10-phenanthroline and EDTA, suggesting that both enzymes were metalloproteinases. The specific fibrinolytic activities of jarar afibrase III and jararafibrase IV were 7.5 +/- 0.4 and 6.5 +/- 1.6 uni ts/mg protein, respectively. The enzymes induced local hemorrhage in t he skin of rats. The minimal hemorrhagic doses of jararafibrase III an d IV were 31.0 and 34.0 mu g/rat, respectively. The enzymes displayed broad substrate specificities like the previously purified jararafibra ses I and II. Jararafibrases III and IV degraded type-IV collagen; gel atin, laminin and fibronectin into smaller fragments. The specific act ivities of jararafibrase III for type-IV collagen and gelatin were 7.6 +/- 0.3 and 43 +/- 11 units/mg protein, respectively. The specific ac tivities of jararafibrase IV for type IV-collagen and gelatin were 16. 5 +/- 1.2 and 1 12 +/- 9 units/ mg protein, respectively.