Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability

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Abstract

In the present study, a novel fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom was purified and characterized in terms of enzyme kinetics and substrate specificity. The purified enzyme [termed snake venom metalloprotease-Macrovipera mauritanica (SVMP-MM)] was composed of a single polypeptide with an apparent molecular weight of 27 kDa, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminus of the enzyme was composed of NH 2 -QRFAPRYIEL-COOH, as determined by N-terminal sequencing. The Aα- and the Bβ-chains of fibrinogen were completely cleaved by SVMP-MM within 20 and 480 min, respectively. However, the γ-chain was much more resistant to digestion by the enzyme. The enzyme also exhibited proteolytic activity, cleaving the α-α polymer of cross-linked fibrin, but did not effectively digest theγ-γ polymer. To determine the kinetic parameters for SVMP-MM, a fluorescence-quenching peptide (termed o-aminobenzoic acid-HTEKLVTS-2,4-dinitrophenyl-NH 2) containing a K-L sequence for SVMP-MM cleavage was designed and synthesized. The optimal pH and temperature for the enzyme activity were found to be 5.5 and 37°C, respectively, when the fluorogenic substrate was synthesized and used as a substrate. Among the various divalent cations tested, Ni 2+ and Cu 2+ showed strong inhibitory effects on enzyme activity, with an average of 69.6% inhibition. The enzyme activity was also inhibited by treatment with 1,10-phenanthroline, ethylenediaminetetraacetic acid and glycol-bis-(2-aminoethylether)-N,N,N′,N′-tetra acetic acid, but not with aprotinin, tosyl-lysine chloromethyl ketone and tosyl-phenylalanyl chloromethyl ketone, suggesting that SVMP-MM is a metalloprotease and not a serine protease. The enzymatic parameters, including the K M, k cat, and k cat /K M values were estimated to be 0.015 mM, 0.031 sec -1, and 20.67 mM -1 sec -1, respectively. SVMP-MM induced vascular permeability by digesting type IV collagen. The results obtained in our study demonstrate that SVMP-MM is a fibrin(ogen)olytic P-I class metalloprotease, which can induce a hemorrhagic reaction in vivo.

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Lee, E. H., Park, J. E., Park, J. W., & Lee, J. S. (2014). Purification and biochemical characterization of a fibrin(ogen)olytic metalloprotease from Macrovipera mauritanica snake venom which induces vascular permeability. International Journal of Molecular Medicine, 34(4), 1180–1190. https://doi.org/10.3892/ijmm.2014.1864

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