PURIFICATION AND CHARACTERIZATION OF A FIBRINOGEN-CLOTTING ENZYME FROM THE VENOM OF JARARACUCU (BOTHROPS-JARARACUSSU)

A dotting enzyme of the venom of Bothrops jararacussu, denoted FC-Bj, was purified by gel chromatography on Sephadex G-100 followed by HPLC on DEAE-5PW-PAK and gel filtration on Sephacryl S-200HR. The enzyme wa s identified as an acidic glycoprotein which probably consists of a si ngle polypeptide chain with isoelectric point values in the range 3.3- 4.4 and containing approx. 19% carbohydrates. On polyacrylamide gel el ectrophoresis (PAGE) at pH 8.3, the enzyme presented a diffuse protein band. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (S DS-PAGE), the enzyme showed two protein bands corresponding to mol. wt s of 50,600 and 60,000. After treatment of the enzyme with neuraminida se, a strongly stained band and a band weaker in staining intensity we re observed on SDS-PAGE, thereby reducing the mel. wts to 44,500 and 5 6,300, respectively. The clotting factor possessed N-alpha-benzoyl-DL- arginine p-nitroanilide hydrolysing activity and coagulated fibrinogen to fibrin. These activities were 0.548 units/mg and 50.55 NIH thrombi n units/mg, respectively. The proteinase was of the serine type, as in dicated by sensitivity to phenylmethanesulfonyl fluoride and benzamidi ne. However, the amidolytic activity of this enzyme was resistant to i nhibitors such as heparin, aprotinin, agmatine, EDTA, I-2581 and TLCK. The importance of disulfide bridges for the structural integrity of t he purified enzyme was indicated by the loss of amidolytic activity in the presence of beta-mercaptoethanol and dithiothreitol. SDS-PAGE of fibrinogen degraded with this enzyme revealed the disappearance of the A alpha and B beta chains and the appearance of lower mel. wt fragmen ts. The enzyme was able to hydrolyse synthetic chromogenic substrates with arginine as the C-terminal residue, and the kinetic parameters we re determined. It hydrolysed the plasma kallikrein substrate H-D-Pro-P he-Arg-pNA (S-2302) and produced kinin-releasing activity causing ileu m contraction, In addition, hypotension and bradycardia were observed in urethane-anesthetized rats upon i.v. injection of the enzyme. Copyr ight (C) 1996 Elsevier Science Ltd