A novel fibrinogen-clotting enzyme, TL-BJ, from the venom of the snake Bothrops jararaca: purification and characterization.
Three chromatographically distinct forms of a novel fibrinogen-clotting serine endopeptidase, TL-BJ1, 2 and 3, were purified from the venom of Bothrops jararaca by a combination of ammonium sulfate precipitation and chromatographic steps. The three forms of TL-BJ have similar amidolytic and plasma coagulating activities. TL-BJ 1, TL-BJ 2 and TL-BJ 3 cause the specific clotting of fibrinogen with release of fibrinopeptide A, the specific activities are 16.8 NIH U/mg (TL-BJ 1), 16.7 NIH U/mg (TL-BJ 2) and 20.8 NIH U/mg (TL-BJ 3). The most sensitive chromogenic substrates for measuring the amidolytic activity of TL-BJ 3 were D-Pro-Phe-Arg-pNA, D-Phe-pipecolyl-Arg-pNA and Z-D-Arg-Gly-Arg-pNA. The amidolytic and coagulant activities of TL-BJ were inhibited by phenylmethylsulfonyl fluoride but not by hirudin. Benzamidine derivatives, which are competitive inhibitors of trypsin-like serine endopeptidases, also inhibited the amidolytic activity of TL-BJ. In SDS/PAGE the main bands of TL-BJ 1, 2 and 3 showed molecular masses of 30 kDa, 31 kDa and 32 kDa. Upon incubation with N-glycosidase F only TL-BJ 3 remained unchanged, whereas TL-BJ 1 and TL-BJ 2 showed products with molecular masses around 23 kDa. Thus, TL-BJ 3 does not seem to be N-glycosylated. The N-terminal amino acid sequences of TL-BJ 2 and TL-BJ 3 are identical while TL-BJ 1 has five substitutions.
Serrano
S M
SM
Laboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, Brazil. s_serrano@hotmail.com
Sampaio
C A
CA
Mentele
R
R
Camargo
A C
AC
Fink
E
E
D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops / Q000235:genetics; D003435:Crotalid Venoms / Q000201:enzymology* / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D003435:Crotalid Venoms; D009842:Oligopeptides; D005340:Fibrinogen; D010450:Endopeptidases; D012697:Serine Endopeptidases