Novel fluorescent substrates for detection of trypsin activity and inhibitor screening by self-quenching.

A group of self-quenching-based substrates with two fluorescent peptides for detection of trypsin activity was designed and synthesized. The substrates could be easily synthesized using simple solid-phase peptide synthesis techniques. Two fluorescent peptide substrates for trypsin were conjugated to the amino groups of lysine as a branched unit. The fluorescence of these substrates was self-quenched owing to the highly assembled fluorophores on the substrates. The release of these concentrated fluorophores by proteases allows for fluorescence recovery. Self-quenching reduced the fluorescence of the substrates by 64.1%, and the fluorescence intensity was recovered by the release of the fluorophores from the substrate peptides via tryptic cleavage. The kinetic assay revealed that the kcat/Km values of the substrates were almost comparable to those of the standard fluorescent probe, peptide-MCA. The detection limit for trypsin was 111pM, and the calculation of IC50 and Ki values for the Bowman-Birk inhibitor was achieved using these substrates. These easily synthesizable self-quenching-based substrates have the potential to be useful for the detection of other disease-related protease activities.