Fluorometric Enzymatic Assay for Proteases

Ten microliters of trypsin/chymotrypsin (Sigma Aldrich, Dorset, UK) working solution (0.1 µM in 1 mM HCl) was added into the wells of a black micro-titre plate with corresponding substrate and peptide replicates (0.1–100 µM) in 10 mM phosphate buffer (final volume 210 μL). Additionally, Phe-Pro-Arg- NHMec (Bachem, Cambridge, UK) and Succinyl-Ala-Ala-Pro-Phe-NHMec (Bachem, Cambridge, UK) were performed as substrates for trypsin and chymotrypsin, respectively. The fluorescence intensity of NHMec was monitored at 37 °C continuously for 30 min by a FLUOstar OPTIMA multi-well plate reader (BMG Labtech, Ortenberg, Germany) at wavelengths of 460 nm for emission and 395 nm for excitation. The inhibition curves of different protease were plotted using the Morrison equation and non-linear regression analysis, which were showed as outlined before [26 (link)].