Mca-RPPGFSAFK(Dnp)-OH Fluorogenic Peptide Substrate

ACE activity was determined following incubation with intramolecularly quenched synthetic ACE specific substrate Mca-RPGFSAFK (Dnp)-OH (R&D systems). In the case of cell lysates, 10 µg of total protein was assayed for activity in a buffer with the following composition: 50 mM MES (4-morpholineethanesulphonic acid), 300 mM NaCl, 10 µm ZnCl2 and 0.01% Triton X-100, pH 6.5. Reaction was initiated by the addition of 5×10−5 M substrate. Where applicable, recombinant enzymes were used at a concentration of 0.01 µg per reaction. The fluorescence measurements were performed in the black microtiter plates (Costar) in a total volume of 100 µl. The plates were read using a fluorescence plate reader SpectraMax M5 (Molecular Devices) at an excitation wavelength 320 nm and emission wavelength 405 nm Fluorescence resulting from the substrate hydrolysis increased with time, and achieved maximum by one h with recombinant enzyme however with cell/tissue lysates the maximum fluorescence was observed by four hours of incubation. Therefore fluorescence recorded at one and four hours of reaction time was taken for calculation of percent enzyme inhibition, when using recombinant enzymes and cell/tissue lysates, respectively. ACE activity was defined as the ACE inhibitor, captopril-sensitive fluorescence, and were expressed as percent inhibition.