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Type: Artigo
Title: Functional and structural characterization of a new serine protease with thrombin-like activity TLBan from Bothrops andianus (Andean Lancehead) snake venom
Author: Valeriano-Zapana, Jose Antonio
Steve Segovia-Cruz, Fernando
Miguel Rojas-Hualpa, Jose
Martins-de-Souza, Daniel
Ponce-Soto, Luis Alberto
Marangoni, Sergio
Abstract: A new serine protease with thrombin-like activity (TLBan) from Bothrops andianus (Andean Lancehead) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. TLBan is a glycoprotein that contains both N-linked carbohydrates and sialic acid in its structure, with Mr similar to 29 kDa under reducing conditions and non-reducing similar to 25 kDa conditions and confirmed by MALDI-TOF mass spectrometry (25,835.65 Da) and exhibited high specificity for BA rho NA, Michaelis-Menten behavior with Km 5.4 x 10(-1) M and the V-max 7.9 x 10(-1) nmoles rho-NA/L/min for this substrate and high stability when was analyzed at different temperatures (25 to 60 degrees C), pHs (4.0 to 8.0), was inhibited by soybean trypsin inhibitor, EDTA and phenylmethylsulfonyl fluoride (PMSF). The total amino acid sequence was obtained through sequencing of selected tryptic peptides and by inference obtained using SwissProt database with the search restricted to serine proteases from Crotalinae snakes and show high amino acid sequence identity with other serine proteases from snake venom. TLBan showed the presence of His(44), Asp(91) residues and Ser was deduced (187) position, in the corresponding positions to the catalytic triad established in the serine proteases and Ser(187) are inhibited by phenylmethylsulfonyl fluoride (PMSF). In this work, we investigated the ability of TLBan to degrade fibrinogen and we observed that it is able to cause alpha- and beta-chain cleavage. Enzymatic activities as well as the platelet aggregation were strongly inhibited when were incubated with PMSF, a specific inhibitor of serine protease. TLBan showed a potential medical-scientific interest to understand the pathophysiological mechanism of the snake venom action and identification of new blood coagulation cascade acting enzymes of natural sources
Subject: Venenos de serpentes
Country: Reino Unido
Editor: Elsevier
Rights: Fechado
Identifier DOI: 10.1016/j.toxicon.2011.11.018
Date Issue: 2012
Appears in Collections:IB - Artigos e Outros Documentos

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