MCA

MCA (7-Methoxycoumarin-4-acetic acid) is a Coumarin (HY-N0709) derivative. MCA quantitates platelet-activating factor (PAF) by high-performance liquid chromatography with fluorescent detection. MCA can modify FRET peptide substrates for analyzing protease activities^[1][2]. In Vitro:1. FRET preparation^[1]
1) Synthesize FRET substrates on TCP resin using Fmoc/tBu chemistry by fully automated solid-phase peptide synthesis.
2) Use a 7-fold molar excess of single Fmoc-L-amino acids for the coupling of standard D- or L-amino acids.
3) Perform Fmoc deprotections twice, each for 10 min, with 30% piperidine in dimethylformamide.
4) Couple Nα-Fmoc-N-ε-2,4-DNP-L-lysine [Fmoc-Lys(Dnp)] in 2-fold excess within 2 h with 1-H-benzotriazolium-1-[bis(dimethylamino) methylene]-5-chloro-tetrafluoro-borate(1-)3-oxide/Oxima Pure.
5) Modify the peptide resin with 7-methoxycoumarin-4-acetic acid (MCA). Carry out the coupling with five equivalents of MCA in dimethylformamide with diisopropylcarbodiimide/Oxima Pure within 3 h and monitor by Kaiser assay.
6) Cleave the FRET substrates off the resin within 4 h by treating the resins with trifluoroacetic acid/phenol/ethanedithiol/thioanisole/water (95:2:1:1:1), and then precipitate and wash them three times with diethylether.
7) Lyophilize the peptides in tert-butyl alcohol/water (4:1).
8) Characterize the FRET substrates analytically by reversed-phase HPLC-electrospray mass spectrometry to ensure the compound purity is greater than 85% (UV 214 nm).
9) Prepare the working solution with the prepared FRET substrates according to experimental requirements. For example, in some experiments, the final concentration of the FRET substrate is prepared to be 0.5 mM (when incubated with proteases).
2. Enzyme activity analysis
1) Produce recombinant catalytically active NSP4 (such as S-tag NSP4).
2) Add NSP4, NE, PR3, or CG at a final concentration of 6 mM to a commercial peptide library containing 0.5 mM FRET substrates in total.
3) Generate fluorescent signals by excitation at 320 nm and record them at 405 nm emission wavelength.
4) Only use the initial linear portion of the progress curve to calculate the enzymatic activity, expressed as relative fluorescence units per minute (increase of relative fluorescence units per minute). Set the value of the substrate that is cleaved best by the tested proteases to 100%, and present the average values with standard deviations as a percentage of this maximum value.
5) Analyze the cleaved fragments by liquid chromatography-tandem mass spectrometry after incubating for 5 and 30 min.