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/Kmvalues 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 IC50and Kivalues 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.
Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0196, Japan.
Daisuke
D
Sato
Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0196, Japan. Electronic address: tmkato@life.kyutech.ac.jp.
Tamaki
T
Kato
D000595:Amino Acid Sequence; D004353:Drug Evaluation, Preclinical / Q000379:methods; D057075:Enzyme Assays / Q000379:methods; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology*
D005456:Fluorescent Dyes; D010455:Peptides; D014361:Trypsin Inhibitors; D014357:Trypsin
Enzyme reaction; Fluorescent peptide; Inhibitor evaluation; Self-quenching substrate; Trypsin detection