Characterization of thrombin like enzyme from Bothrops pictus venom

Authors

  • Dan Vivas-Ruiz Laboratorio de Biologia Molecular. Facultad de Ciencias Biologicas. Universidad Nacional Mayor de San Marcos. Lima Peru
  • Gustavo A. Sandoval Laboratorio de Biologia Molecular. Facultad de Ciencias Biologicas. Universidad Nacional Mayor de San Marcos. Lima Peru.
  • Fanny Leo Laboratorio de Biologia Molecular. Facultad de Ciencias Biologicas. Universidad Nacional Mayor de San Marcos. Lima Peru.
  • Edith Rodríguez Laboratorio de Biologia Molecular. Facultad de Ciencias Biologicas. Universidad Nacional Mayor de San Marcos. Lima Peru.
  • Armando Yarlequé Laboratorio de Biologia Molecular. Facultad de Ciencias Biologicas. Universidad Nacional Mayor de San Marcos. Lima Peru.
  • Eladio Flores-Sánchez Laboratorio de Bioquimica y Quimica de Proteinas, Fundacion Ezequiel Dias. Bello Horizonte Brasil.

DOI:

https://doi.org/10.17843/rpmesp.2015.324.1754

Keywords:

Bothrops, Thrombin, Blood coagulation, Fibrinogen

Abstract

Objectives. To perform a biochemical and molecular characterization of the coagulant principle from Bothrops pictus venom. Materials and 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.

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Published

2015-12-05

Issue

Section

Original Article

How to Cite

1.
Vivas-Ruiz D, Sandoval GA, Leo F, Rodríguez E, Yarlequé A, Flores-Sánchez E. Characterization of thrombin like enzyme from Bothrops pictus venom. Rev Peru Med Exp Salud Publica [Internet]. 2015 Dec. 5 [cited 2024 Apr. 19];32(4):652-8. Available from: https://rpmesp.ins.gob.pe/index.php/rpmesp/article/view/1754

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