Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom

• DOI: 10.1016/j.toxicon.2007.11.017
• Publication Date: 2007-11-26T17:09:02Z
• Authors: Carolina P. Berna...; Norival A. Santos...; Tássia R. Costa; Mário S.R. Gomes; Fernanda S. Torres; Júnia Costa; Márcia H. Borges; Michael Richardson; Daniel M. dos Santos; Adriano M. de Cas...; Maria I. Homsi-Br...; Andreimar M. Soares; Fábio de Oliveira
• Keywords: 0303 health sciences; 03 medical and health sciences; Base Sequence; Crotalid Venoms; Molecular Sequence Data; Metalloproteases; Animals; Fibrinogen; Bothrops; Amino Acid Sequence; Cloning, Molecular; 3. Good health

A proteinase, named BmooMPalpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the Aalpha-chain of fibrinogen first, followed by the Bbeta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMPalpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMPalpha-I activity. Since the BmooMPalpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.