Phe-Pro-Arg-AMC (Bachem, Merseyside, UK) and succinyl-Ala-Ala-Pro-Phe-AMC (Bachem, Merseyside, UK) were used as substrates for trypsin and chymotrypsin inhibition determinations, respectively. Serial peptide dilutions (0.1–100 µM in phosphate buffered saline (PBS)) were incubated with 180 µL of substrate solution (50 µM in PBS) in a black 96-well plate under dark conditions for 10 min. Immediately prior to measurement, 10 µL of trypsin/chymotrypsin working solution (0.1 µM in PBS) was added to each well (final volume of 210 µL) in the plate. Subsequently, the fluorescence intensity was measured with the FLUO star OPTIMA plate reader (BMG Labtech, Ortenberg, Germany). The whole measurement process lasted for 30 min and was carried out at an emission wavelength of 460 nm and an excitation wavelength of 395 nm. The inhibition curves of trypsin and chymotrypsin were analyzed by non-linear regression using the Morrison equation in Prism 9 (Trypsin Km = 67.38 μM; Chymotrypsin Km = 17.15 μM; [S] = 42.86 μM; Et = 0.0020 μM).
Phe pro arg amc
Manufactured by Bachem
Sourced in United Kingdom
Phe-Pro-Arg-AMC is a synthetic peptide substrate used in biochemical assays. It contains the amino acid sequence phenylalanine-proline-arginine and is conjugated to the fluorogenic reporter molecule 7-amino-4-methylcoumarin (AMC). The cleavage of the peptide bond releases the AMC moiety, which can be detected by fluorescence spectroscopy.
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4 protocols using phe pro arg amc
1
Kinetic Inhibition Assay for Trypsin and Chymotrypsin
2
Enzymatic Inhibition Assays for Proteases
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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)
3
Peptide Inhibition of Trypsin and Chymotrypsin
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The inhibitory activity of peptides towards trypsin and chymotrypsin was tested the same as detailed in Wang’s study [11 (link)]. Peptides were initially prepared in a range of 10−3–10−6 M and then diluted to 16 concentrations. Each dilution was tested in duplicates. Additionally, Phe-Pro-Arg-AMC and Succinyl-Ala-Ala-Pro-Phe-NHMec (obtained from Bachem, UK) were served as substrates for trypsin and chymotrypsin, respectively.
4
Trypsin Inhibitory Activity Assay
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Peptide was dissolved to make a stock solution at 1 × 104 μM with phosphate-buffered saline (PBS), and then diluted to a series of working concentrations ranging from 1 to 1000 μM. To each well of a black 96-well plate, a working peptide concentration, 180 μL of substrate (50 μM in PBS), and 10 μL of trypsin (0.1 μM in 1 mM HCl) were added up to a final volume of 210 μL in a dark environment. The trypsin was added last, and the plate was measured immediately. The fluorescence intensity of each well was recorded for 30 min at every 30 s using a FLUOstar OPTIMA plate reader (BMG Labtech, Ortenberg, Germany). As the substrate of trypsin in this assay was Phe-Pro-Arg-AMC (Bachem, Merseyside, UK), the emission and excitation wavelengths were 460 nm and 395 nm, respectively, and were measured at 37 °C. The curves of the trypsin inhibitory activity were generated using the Morrison equation in Prism 9 (the working concentration of the substrate, [S] = 42.86 μM; the working concentration of trypsin, Et = 0.0020 μM; trypsin kinetic constant, Km = 41.07 μM).
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