Isolation and biological characterization of Batx-I, a weak hemorrhagic and fibrinogenolytic PI metalloproteinase from Colombian Bothrops atrox venom.
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 microg/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 A alpha- and B beta-chains, although partial degradation of the gamma-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.
Patiño
Arley C
AC
Programa de Ofidismo/Escorpionismo, Universidad de Antioquia, Medellín 1226, Colombia. arleycamilo@gmail.com
Pereañez
Jaime A
JA
Núñez
Vitelbina
V
Benjumea
Dora M
DM
Fernandez
Maritza
M
Rucavado
Alexandra
A
Sanz
Libia
L
Calvete
Juan J
JJ
D000818:Animals; D017837:Bothrops / Q000502:physiology*; D002364:Caseins / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003105:Colombia; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D004591:Electrophoresis, Polyacrylamide Gel; D005342:Fibrinolysis / Q000187:drug effects*; D006470:Hemorrhage / Q000139:chemically induced*; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000633:toxicity*; D018482:Muscle, Skeletal / Q000201:enzymology / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization
D002364:Caseins; D003435:Crotalid Venoms; D011480:Protease Inhibitors; C026018:azocasein; C554059:Batx-I, Bothrops atrox; D045726:Metalloproteases