| Assay Method Information | |
| | MMP Activity Assay |
| Description: | The assay was carried out in white NUNC 96-well plates as previously described [Puerta et al., J. Biol. Inorg. Chem., 11:131-138]. Each well contained a total volume of 90 μl including buffer (50 mM HEPES, 10 mM CaCl2, 0.05% Brij-35, pH 7.5), human recombinant MMP (1.16 U/well for MMP2 and 0.007 U/well for MMP12), and the fragment solution. This mixture was incubated at 37 °C for 30 min. The reaction was then initiated by the addition of 10 μl of the fluorogenic MMP substrate (4 μM final concentration, Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2-AcOH) (Mca = (7-methoxycoumarin-4-yl) acetyl; Dpa = N-3-(2,4-dinitrophenyl)-L-a,β-diaminopropionyl). Fluorescence measurements were recorded using a BioTek Synergy H4 fluorescence plate reader every minute for 20 min with excitation and emission wavelengths at 320 and 400 nm, respectively. |
| Affinity data for this assay | |
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