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

The present study aimed to evaluate the proteolytic and biological activities of a new metalloproteinase from B. moojeni venom. The purification of BmooMP α -II was carried out through two chromatographic steps (ion-exchange and affinity). BmooMP α -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. BmooMP α -II cleaves A α -chain of fibrinogen followed by B ß -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 BmooMP α -II is a metalloproteinase. This proteinase was devoid of haemorrhagic, coagulant, or anticoagulant activities. BmooMP α -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 BmooMP α -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.

Isolation and characterization of moojenin, an acid-active, anticoagulant metalloproteinase from Bothrops moojeni venom.

A fibrinogenolytic metalloproteinase from Bothrops moojeni venom, named moojenin, was purified by a combination of ion-exchange chromatography on DEAE-Sephacel and gel filtration on Sephacryl S-300. SDS-PAGE analysis indicated that moojenin consists of a single polypeptide chain and has a molecular mass about 45 kDa. Sequencing of moojenin by Edman degradation revealed the amino acid sequence LGPDIVSPPVCGNELLEVGEECDCGTPENCQNE, which showed strong identity with many other snake venom metalloproteinases (SVMPs). The enzyme cleaves the Aα-chain of fibrinogen first, followed by the Bß-chain, and shows no effects on the γ-chain. Moojenin showed a coagulant activity on bovine plasma about 3.1 fold lower than crude venom. The fibrinogenolytic and coagulant activities of the moojenin were abolished by preincubation with EDTA, 1,10-phenanthroline and ß-mercaptoethanol. Moojenin showed maximum activity at temperatures ranging from 30 to 40 °C and its optimal pH was 4.0. Its activity was completely lost at temperatures above 50 °C. Moojenin induced necrosis in liver and muscle, evidenced by morphological alterations, but did not cause histological alterations in mouse lungs, kidney or heart. Moojenin rendered the blood uncoagulatable when it was intraperitoneally administered into mice. This metalloproteinase may be of medical interest because of its anticoagulant activity.