Boc phe ser arg nhmec

Trypsin and chymotrypsin inhibition assays were performed as previously described [24 (link)]. The substrate of trypsin and chymotrypsin were Phe-Pro-Arg-AMC and Succinyl-Ala-Ala-Pro-Phe-NHMec, respectively, and obtained from Bachem, Merseyside, UK. In the tryptase inhibition assay, peptide samples were first dissolved 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) to obtain different concentrations of 0.5, 1, 2 and 4 mM and then added to the wells of a black 96-well plate containing (Boc-Phe-Ser-Arg-NHMec, obtained from Bachem, Merseyside, UK) (50 μM). Before measurement, tryptase (2.5 μL from 1 mg/mL stock solution, Calbiochem, Nottingham, UK) was added to each well. The rate of hydrolysis of substrate was monitored continuously at 37 °C and 460 nm with a Fluostar Optima plate reader (BMG Labtech GmbH, Offenburg, Germany)

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.

The trypsin, chymotrypsin inhibition tests were performed as described previously [22 (link)]. 10 µL tryptase (1 mg/mL, Calbiochem, UK) was added to the wells of a micro-titer plate containing 180 µL substrate (Boc-Phe-Ser-Arg-NHMec, obtained from Bachem, UK) (50 µM) and 20 µL synthetic replicates (0.1–100 µM) in a tryptase buffer, pH7.6, containing 0.05 M Tris, 0.15 M NaCl, and 0.2% (w/v) polyethylene glycol 6000 (final volume 210 µL).
The rate of hydrolysis of the substrate was monitored by measuring the rate of increase of fluorescence due to the release of 7–amino–4–methylcoumarin (AMC) at 460 nm (excitation 360 nm) in a FLUOstar OPTIMA multi-well plate reader. The inhibition curves of the trypsin/chymotrypsin inhibition assay and tryptase inhibition assay were formed as outlined before [11 (link),12 (link)].