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

• Published in: BioMed research international Vol. 2014; no. 2014; pp. 1 - 12
• Main Authors: Ribeiro de Queiroz, Mayara, Mamede, Carla Cristine N., Cortes Fonseca, Kelly, de Morais, Nadia Cristina G., Barbosa de Sousa, Bruna, Santos-Filho, Norival A., Beletti, Marcelo E., Arantes, Eliane C., Stanziola, Leonilda, de Oliveira, Fábio
• Format: Journal Article
• Language: English
• Published: Cairo, Egypt Hindawi Puplishing Corporation 01-01-2014; Hindawi Publishing Corporation; John Wiley & Sons, Inc; Hindawi Limited
• Subjects: Amino Acid Sequence; Animals; Binding sites; Blood platelets; Bothrops - metabolism; Bothrops moojeni; Cattle; Collagen; Colleges & universities; Crotalid Venoms - enzymology; Enzymes; Experiments; Fibrinogen; Fibrinogen - metabolism; Humans; Laboratories; Male; Metalloenzymes; Metalloproteases - chemistry; Metalloproteases - isolation & purification; Metalloproteases - pharmacology; Mice; Molecular Sequence Data; Molecular weight; Necrosis; Organ Specificity - drug effects; Platelet Aggregation - drug effects; Platelet Aggregation Inhibitors - chemistry; Platelet Aggregation Inhibitors - isolation & purification; Platelet Aggregation Inhibitors - pharmacology; Proteins; Proteolysis - drug effects; Rats, Wistar; Sequence Alignment; Venom; Brazil
• ISSN: 2314-6133; 2314-6141
• DOI: 10.1155/2014/352420

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.