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 on CM Sepharose. The enzyme called leucurolysin-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/microg, respectively; crude venom=8.0 units/mg and 9.5 units/microg). 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 Ala14-Leu15 and Tyr16-Leu17 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 microg) subcutaneously into mice.
Bello
C A
CA
Research and Development Center, Ezequiel Dias Fundation, 30510-010 Belo Horizonte, Brazil.
Hermogenes
A L N
AL
Magalhaes
A
A
Veiga
S S
SS
Gremski
L H
LH
Richardson
M
M
Sanchez
Eladio F
EF
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D017837:Bothrops; D003435:Crotalid Venoms / Q000201:enzymology* / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D007797:Laminin / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D003435:Crotalid Venoms; D005353:Fibronectins; D007797:Laminin; D008562:Membrane Glycoproteins; C029967:nidogen; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D010450:Endopeptidases