Purification and characterization of a fibrinogen-clotting enzyme from the venom of jararacuçu ( Bothrops jararacussu)

Abstract

A clotting 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 was identified as an acidic glycoprotein which probably consists of a single polypeptide chain with isoelectric point values in the range 3.3–4.4 and containing approx. 19% carbohydrates. On polyacrylamide gel electrophoresis (PAGE) at pH 8.3, the enzyme presented a diffuse protein band. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the enzyme showed two protein bands corresponding to mol. wts of 50,600 and 60,000. After treatment of the enzyme with neuraminidase, a strongly stained band and a band weaker in staining intensity were observed on SDS-PAGE, thereby reducing the mol. wts to 44,500 and 56,300, respectively. The clotting factor possessed N- α-benzoyl-DL-arginine p-nitroanilide hydrolysing activity and coagulated fibrinogen to fibrin. These activities were 0.548 units/mg and 50.55 NIH thrombin units/mg, respectively. The proteinase was of the serine type, as indicated by sensitivity to phenylmethanesulfonyl fluoride and benzamidine. However, the amidolytic activity of this enzyme was resistant to inhibitors such as heparin, aprotinin, agmatine, EDTA, I-2581 and TLCK. The importance of disulfide bridges for the structural integrity of the purified enzyme was indicated by the loss of amidolytic activity in the presence of β-mercaptoethanol and dithiothreitol. SDS-PAGE of fibrinogen degraded with this enzyme revealed the disappearance of the Aα and Bβ chains and the appearance of lower mol. wt fragments. The enzyme was able to hydrolyse synthetic chromogenic substrates with arginine as the C-terminal residue, and the kinetic parameters were determined. It hydrolysed the plasma kallikrein substrate H-D-Pro-Phe-Arg-pNA (S-2302) and produced kinin-releasing activity causing ileum contraction. In addition, hypotension and bradycardia were observed in urethane-anesthetized rats upon i.v. injection of the enzyme.