Isolation and biological characterization of Batx-I, a weak hemorrhagic and fibrinogenolytic PI metalloproteinase from Colombian Bothrops atrox venom |
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Authors: | Arley C. Patiñ o,Jaime A. Pereañ ez,Dora M. Benjumea,Alexandra Rucavado,Juan J. Calvete |
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Affiliation: | a Programa de Ofidismo/Escorpionismo, Universidad de Antioquia, Medellín 1226, Colombia b Escuela de Microbiología, Universidad de Antioquia, Medellín 1226, Colombia c Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica d Instituto de Biomedicina de Valencia, CSIC, Valencia, Spain |
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Abstract: | A hemorrhagic metalloproteinase, named Batx-I, was isolated from the venom of Bothrops atrox specimens (from Southeastern Colombian region) by a combination of CM-Sephadex C25 ion-exchange and Affi-gel Blue affinity chromatographies. This enzyme accounts for about 45% of venom proteins, and it has an ESI-MS isotope-averaged molecular mass of 23296.2 Da and a blocked N-terminus. Two internal fragments sequenced by mass spectrometric analysis showed similarity to other SVMPs from Bothrops venoms. To investigate the possible participation of Batx-I in the envenomation pathophysiology, proteolytic, fibrinogenolytic, hemorrhagic, and other biological activities were evaluated. The minimal hemorrhagic dose obtained was 17 μg/20 g body weight. The enzyme showed proteolytic activity on azocasein, comparable with activity of BaP1. This activity was inhibited by EDTA and 1, 10 o-phenanthroline but not by aprotinin, pepstatin A or PMSF. Fibrinogenolytic activity was analyzed by SDS-PAGE, revealing a preference for degrading the Aα- and Bβ-chains, although partial degradation of the γ-chain was also detected. The protein lacks coagulant and defibrinating activity. The CK levels obtained, clearly reflects a myotoxic activity induced by Batx-I. The hemorrhagic and fibrinogenolytic activities exhibited by the isolated PI-SVMP may play a role in the hemorrhagic and blood-clotting disorders observed in patients bitten by B. atrox in Colombia. |
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Keywords: | Bothrops atrox PI metalloproteinase Fibrinogenolytic activity Hemorrhagic activity Envenomation pathophysiology |
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