Phe pro arg nhmec

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)].

Trypsin (10 μl from 0.1 μM stock solution in 1 mM HCl), was added to the wells of a microtitre plate containing substrate (Phe-Pro-Arg-NHMec) (50 μM) and synthetic peptide replicates (0.1–100 μM) in 10 mM phosphate buffer, pH 7.4, containing 2.7 mM KCl and 137 mM NaCl (final volume 210 μl).
Chymotrypsin (10 μl from 0.1 μM stock solution in 1 mM HCl) was added to the wells of a microtitre plate containing substrate (Succinyl–Ala–Ala–Pro–Phe–NHMec, obtained from Bachem, U.K.) (50 μM) and synthetic peptide replicates (0.1–100 μM) in 10 mM phosphate buffer, pH 7.4, containing 2.7 mM KCl and 137 mM NaCl (final volume 210 μl).
Tryptase (2.5 μl from 1 mg/ml stock solution, Calbiochem, U.K.), was added to the wells of a microtitre plate containing substrate (Boc-Phe-Ser-Arg-NHMec, obtained from Bachem, U.K.) (50 μM) and synthetic peptide replicates (0.5, 1, 2 and 4 mM) in tryptase assay buffer, pH 7.6, containing 0.05 M Tris, 0.15 M NaCl and 0.2% (w/v) PEG 6000 (final volume 210 μl).
Each determination was carried out in triplicate. The rate of hydrolysis of substrate was monitored continuously, at 37°C, by measuring the rate of increase in fluorescence due to production of 7-amino-4-methylcoumarin (NH₂Mec) at 460 nm (excitation 360 nm) in a CytoFluor® multi-well plate reader Series 4000 spectrofluorimeter.