Table of Contents
Journal of Toxins
Volume 2013, Article ID 207170, 11 pages
Research Article

Biochemical and Pharmacological Characterization of TLBbar, a New Serine Protease with Coagulant Activity from Bothrops barnetti Snake Venom

Department of Biochemistry, Institute of Biology (IB), State University of Campinas (UNICAMP), P.O. Box 6109, 13083-970 Campinas, SP, Brazil

Received 4 April 2013; Accepted 6 June 2013

Academic Editor: Andreimar Martins Soares

Copyright © 2013 Magaly Alejandra Brousett-Minaya et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


A thrombin-like enzyme named TLBbar was isolated from Bothrops barnetti snake venom and its biochemical and pharmacological characteristics were determined. TLBbar was purified using size exclusion chromatography and reverse phase HPLC, showing molecular mass of 28750.7 Da determined by mass spectrometry. TLBbar serine protease is basic (pI 7.4) and its structure shows similarity with other serine proteases of snake venom. Optimal proteolytic activity was at 37°C and pH 8; this activity was strongly inhibited by PMSF and Leupeptin, however; heparin, and soybean trypsin inhibitor (SBT-I) were ineffective. Kinetic studies on BApNA chromogenic substrate have revealed that TLBbar presents a Michaelis-Menten kinetics, with values of and of 0.433 mM and 0.42 nmol/min, respectively. TLBbar showed high clotting activity upon bovine and human plasma, presenting IC of 125 and minimum dose coagulant (MDC) of 2.23 μg/μL. TLBbar cleavages the Aα chain of bovine fibrinogen, with maximal efficiency at 30–40°C in the presence of calcium after two hours incubation; this fibronogenolityc activity was inhibited by PMSF and Leupeptin, confirming its classification in the group of serine proteases. In addition, TLBbar is capable of aggregating platelets in the same way that thrombin in concentrations of 2.5 μg/μL.