Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom

In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed-jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography oil CM Sepharose. The enzyme called leucrolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH2-terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity = 21.6 units/mg and 17.5 units/Pg, respectively; Crude venom = 8.0 units/mg and 9.5 units/mu g). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a. cleaved the Ala(14)-Leu(15) and Tyr(16)-Leu(17) bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the Purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from Purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 mu g) Subcutaneously into mice. (c) 2005 Elsevier SAS. All rights reserved.