Please use this identifier to cite or link to this item: http://repositorio.unicamp.br/jspui/handle/REPOSIP/200395
Type: Artigo de periódico
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, José Antonio
Segovia-Cruz, Fernando Steve
Rojas-Hualpa, José Miguel
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 ∼29 kDa under reducing conditions and non-reducing ∼25 kDa conditions and confirmed by MALDI-TOF mass spectrometry (25,835.65 Da) and exhibited high specificity for BAρNA, Michaelis-Menten behavior with Km 5.4 × 10(-1) M and the V(max) 7.9 × 10(-1) nmoles ρ-NA/L/min for this substrate and high stability when was analyzed at different temperatures (25 to 60 °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 http://br.expasy.org/ 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 α- and β-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: Amino Acid Sequence
Animals
Blood Coagulation
Bothrops
Crotalid Venoms
Fibrinogen
Male
Mass Spectrometry
Mice
Molecular Sequence Data
Platelet Aggregation
Rats
Rats, Sprague-dawley
Sequence Homology, Amino Acid
Serine Proteases
Thrombin
Rights: fechado
Identifier DOI: 10.1016/j.toxicon.2011.11.018
Address: http://www.ncbi.nlm.nih.gov/pubmed/22155303
Date Issue: 2012
Appears in Collections:Artigos e Materiais de Revistas Científicas - Unicamp

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