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

DOI: 10.1155/2013/207170

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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. 1. Introduction The snake venom contains a variety of proteins that are studied in the world for biological and pharmacological importance; within their complex composition has proteolytic enzymes which belong to two groups: serine proteases and metalloproteases. Both groups affect the hemostatic system through several mechanisms [1, 2]. Over 20 serine proteases families (named S1 to S66) have been identified so far and they are grouped according to their functional and structural similarity. Snake venom serine proteases belong to trypsin S1 family clan SA. To date, a large number of studies, including molecular cloning, have led to the isolation and identification of TLEs mainly from the venom of subfamily Viperinaeand Crotalinae [3–9]. These enzymes have a common catalytic mechanism, which includes high reactivity of the serine residue that has an important role in the formation of the transient acyl-enzyme complex, which is stabilized by the presence of histidine residues and aspartic acid within the active site. The amino acids involved in this mechanism correspond to the catalytic triad (Ser 195, His 57, and Asp 102) that is highly conserved [10]. Serine proteases from snake venom (SVSPs) generally have 12 cysteine residues, 10 of which form five disulfide

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