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 ser ine endopeptidase. TL-BJ 1, 2 and 3, were purified from the venom of Bothro ps jararaca by a combination of ammonium sulfate precipitation and chromato graphic 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 clo tting of fibrinogen with release of fibrinopeptide A. the specific activiti es art: 16.8 NIH U/mg (TL-BJ 1), 16.7 NIH U/mg (TL-BJ 2) and 20.8 NLH U/mg (TL-BJ 3). The most sensitive chromogenic substrates for measuring the amid olytic 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 w ere inhibited by phenylmethylsulfonyl fluoride but not by hirudin. Benzamid ine derivatives, which are competitive inhibitors of trypsin-like serine en dopeptidases, 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 kD a and 32 kDa. Upon incubation with N-glycosidase F only TL-BJ 3 remained un changed, 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-termi nal amino acid sequences of TL-BJ 3 and TL-BJ 3 are identical while TL-BJ 1 has five substitutions.