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| | enzyme-based fluorimetric assay |
| Description: | For this assay, the reagents used as follows. The substrate corresponds to Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg 25 mm in assay buffer. This low-fluorescent peptide is cleaved by MMPs to provide Mca-Pro-Leu-Gly, which is intensely fluorescent at an excitation at 340 nm and emission at 405 nm. While the enzymes pro-MMP-2 250 pm and MMP-9 (recombinant catalytic sequence) 500 pm in assay buffer. The assay buffer consists of Tris HCl 0.1 m, pH 7.5, NaCl 0.1 m, CaCl2 10 mm, 0.05% v/v BRIJ35, APMA (p-aminophenylmercuric acetate) 1 mm. This was carried out in 96-well plate, a solution of inhibitor (50 mL) at the appropriate concentration and enzyme (50 mL) was incubated together at 25 °C for 30 min, and then 10 mL of substrate solution was added and the plate was further incubated 3 h at 37 °C. The incubation solutions were quenched with acetic acid (100 mL of 3% soln.) and their fluorescence was recorded with the help of a plate reader spectrometer. The inhibitory activities were calculated against a control and IC50 curves, derived from 6 different inhibitor concentrations, extrapolated by means of statistical analysis program graphpad prism (v. 4.0; San Diego, CA, USA). Each inhibitor concentration was used in triplicate and the experiments run twice. |
| Affinity data for this assay | |
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