Rapid purification of a new P-I class metalloproteinase from Bothrops moojeni venom with antiplatelet activity.

Mayara R De Queiroz,Norival A Santos-Filho,Kelly C Fonseca,Eliane C Arantes,Carla C Neves Mamede,Nadia C G De Morais,Marcelo E Beletti,Leonilda Stanziola,Fábio De Oliveira,Bruna B De Sousa

doi:10.1155/2014/352420

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.

Highlights

Snake venoms comprise a complex mixture of proteins, organic compounds with low molecular mass, and inorganic compounds [1,2,3]
Proteolytic enzymes from snake venoms have attracted the interest of researchers due to their important role in envenomation caused by Bothrops snakes
We describe the purification and characterisation of a P-I Snake Venom Metalloproteinases (SVMPs) from B. moojeni venom

Summary

Introduction

Snake venoms comprise a complex mixture of proteins, organic compounds with low molecular mass, and inorganic compounds [1,2,3]. Viperid venoms contain powerful enzymatic and nonenzymatic components that affect haemostatic mechanism, such as proteinases, phospholipases A2, and disintegrins [5,6,7,8,9,10,11,12,13,14]. Snake Venom Metalloproteinases (SVMPs) play a key role in local tissue damage and systemic alterations resulting from viperid snake envenomations. These enzymes can induce haemorrhage, necrosis, oedema, skin damage, and inflammation, and degrade extracellular matrix components and impair the regeneration of affected skeletal muscle [6, 9, 15,16,17]. SVMPs can affect platelet function through specific structural or enzymatic effects on platelet receptors or their ligands and the coagulation cascade by multiple mechanisms [2, 13, 15, 18]

Methods

Results

Conclusion