Mca plaqav dpa rsssr nh2

To determine the inhibition of gelatinolytic activity, we used a previously optimized gelatin degradation assay [29 (link)]. Activated MMP-9, activated MMP-2, neutrophil elastase or supernatants of stimulated neutrophils, were incubated with different concentrations of inhibitors and incubated for 30 min at 37 °C. Next, dye-quenched gelatin (DQ™-gelatin, Invitrogen, Carlsbad, CA, USA) was added at a final concentration of 2.5 µg/mL and the increase in fluorescence over time was measured with the CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany). For other MMPs, the OmniMMP substrate peptide (Mca-PLGL-Dpa-AR-NH₂, cat. no. BML-P126-0001, Enzo Life Sciences, Farmingdale, NY, USA) was used at a final concentration of 2.5 µg/mL. For ADAM17 the fluorogenic peptide Mca-PLAQAV-Dpa-RSSSR-NH₂ (ES003, R & D systems, Minneapolis, MO, USA) and for neutrophil elastase the fluorogenic substrate MeOSuc-AAPV-AMC (cat. no. 324740, Merck Millipore, Darmstadt, Germany) were used. Proteolytic activity was determined by linear regression of the fluorescence curve and the percentage inhibition was calculated through comparison with the positive control (no inhibitors). When testing supernatants from human neutrophils, the assay was performed in the presence of 20 µM neutrophil elastase inhibitor (Elastase Inhibitor IV, Calbiochem, Darmstadt, Germany).