Mmp 13

The catalytic domains of MMP-2, MMP-8 and MMP-13 were purchased from Enzo Life Sciences (Lörrach, Germany). The assays were performed in triplicate in 96-well white microtiter plates (Corning, NBS; Corning, NY, USA). For assay measurements, inhibitor stock solutions (DMSO, 10 mM) were diluted to six different concentrations (1 nM–100 µM) in fluorometric assay buffer (50 mM Tris·HCl pH 7.5, 200 mM NaCl, 1 mM CaCl₂, 1 µM ZnCl₂, 0.05% Brij-35 and 1% DMSO). Enzyme and inhibitor solutions were incubated in the assay buffer for 15 min at room temperature before the addition of the fluorogenic substrate solution (OmniMMP^® = Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂, Enzo Life Sciences, 2.5 µM final concentration or OmniMMP^®RED = TQ3-GABA-Pro-Cha-Abu-Smc-His-Ala-Dab(6’-TAMRA)-Ala-Lys-NH₂, (TAMRA: tetramethylrhodamine) Enzo Life Sciences, 1 µM final concentration). After further incubation for 2–4 h at 37 °C, fluorescence was measured (λ_ex = 340 nm, λ_em = 405 nm or λ_ex = 545 nm, λ_em = 572 nm) using a Perkin-Elmer Victor V3 plate reader (Waltham, MA, USA). Control wells lacked inhibitor. The MMP inhibition activity was expressed in relative fluorescence units (RFU). Percent inhibition was calculated from control reactions without inhibitor; IC₅₀ values were determined using GraphPad Prism version 5.0f and are expressed as the mean ± SEM of at least three independent measurements in triplicate.