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BioMed Research International
Volume 2014, Article ID 352420, 12 pages
http://dx.doi.org/10.1155/2014/352420
Research Article

Rapid Purification of a New P-I Class Metalloproteinase from Bothrops moojeni Venom with Antiplatelet Activity

1Instituto de Genética e Bioquímica, Universidade Federal de Uberlândia, 38400-902 Uberlândia, MG, Brazil
2Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica (N-Biofar), 31270-901 Belo Horizonte, MG, Brazil
3Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, SP, Brazil
4Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, 38400-902 Uberlândia, MG, Brazil

Received 24 February 2014; Revised 1 May 2014; Accepted 12 May 2014; Published 1 June 2014

Academic Editor: Jozef Anné

Copyright © 2014 Mayara R. de Queiroz et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The present study aimed to evaluate the proteolytic and biological activities of a new metalloproteinase from B. moojeni venom. The purification of BmooMPα-II was carried out through two chromatographic steps (ion-exchange and affinity). BmooMPα-II is a monomeric protein with an apparent molecular mass of 22.5 kDa on SDS-PAGE 14% under nonreducing conditions. The N-terminal sequence (FSPRYIELVVVADHGMFTKYKSNLN) revealed homology with other snake venom metalloproteinases, mainly among P-I class. BmooMPα-II cleaves Aα-chain of fibrinogen followed by Bβ-chain, and does not show any effect on the γ-chain. Its optimum temperature and pH for the fibrinogenolytic activity were 30–50°C and pH 8, respectively. The inhibitory effects of EDTA and 1,10-phenantroline on the fibrinogenolytic activity suggest that BmooMPα-II is a metalloproteinase. This proteinase was devoid of haemorrhagic, coagulant, or anticoagulant activities. BmooMPα-II caused morphological alterations in liver, lung, kidney, and muscle of Swiss mice. The enzymatically active protein yet inhibited collagen, ADP, and ristocetin-induced platelet aggregation in a concentration-dependent manner. Our results suggest that BmooMPα-II contributes to the toxic effect of the envenomation and that more investigations to elucidate the mechanisms of inhibition of platelet aggregation may contribute to the studies of snake venom on thrombotic disorders.