title: [Characterization of thrombin like enzyme FROM Bothrops pictus venom].
abstract: OBJECTIVE
  To perform a biochemical and molecular characterization of the coagulant principle from Bothrops pictus venom.
  
  
  METHODS
  We amplified the genetic sequence of this enzyme from cDNA and analyzed the homology of its nucleotide sequence and its deduced protein. This enzyme was also purified for N-terminal sequencing of first 20 amino acids and for coagulation assays using human plasma and human fibrinogen. Furthermore, cleavage pattern on fibrinogen was evaluated using SDS-PAGE and defibrinogenant activity on white mice (18-22 g). Finally, associated carbohydrate content, effect of protease inhibitors and chloride ions on its enzymatic activity were analyzed.
  
  
  RESULTS
  The Thrombin-like Enzyme from Bothrops pictus showed homology at primary level of structure with other previously reported TLEs from Viperidae family. Minimum Coagulant Dosis (MCD) on plasma and human fibrinogen were 18 and 6 µg, respectively, and its coagulant potency was 131.1 NHI Thrombin units. This TLE was stable under physiological conditions and chloride ions are not necessary for its activity. Detected associated carbohydrates were hexoses (25.76%), hexosamines (13.12%) and sialic acid (0.76%). Phenyl methyl sulphonyl fluoride (PMSF) and dithiothreitol (DTT) were the main inhibitors of its enzymatic activity, but heparin had no inhibitor effect.
  
  
  CONCLUSIONS
  The coagulant principle of Bothrops pictus venom is a Thrombin-like enzyme.
authors.affiliation: Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú
authors.forename: Dan
authors.initials: D
authors.lastname: Vivas-Ruiz
authors.affiliation: Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú
authors.forename: Gustavo A
authors.initials: GA
authors.lastname: Sandoval
authors.affiliation: Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú
authors.forename: Fanny
authors.initials: F
authors.lastname: Lazo
authors.affiliation: Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú
authors.forename: Edith
authors.initials: E
authors.lastname: Rodríguez
authors.affiliation: Laboratorio de Biología Molecular, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú
authors.forename: Armando
authors.initials: A
authors.lastname: Yarlequé
authors.affiliation: Laboratorio de Bioquímica y Química de Proteínas, Fundación Ezequiel Días, Bello Horizonte Brasil, Bello Horizonte, Brasil.
authors.forename: Eladio
authors.initials: E
authors.lastname: Flores-Sánchez
mesh_terms: D000818:Animals; D017837:Bothrops; D003435:Crotalid Venoms / Q000737:chemistry* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen; D006801:Humans; D051379:Mice; D013917:Thrombin / Q000737:chemistry*
chemical_list: D003435:Crotalid Venoms; D005340:Fibrinogen; D013917:Thrombin