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A real‐time fluorescence assay for protease activity and inhibitor screening based on the aggregation‐caused quenching of a perylene probe
Author(s) -
Wang Yan,
Zhang Zhifang,
Zhang Ya,
Yu Cong
Publication year - 2018
Publication title -
luminescence
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.428
H-Index - 45
eISSN - 1522-7243
pISSN - 1522-7235
DOI - 10.1002/bio.3478
Subject(s) - perylene , protease , fluorescence , chemistry , quenching (fluorescence) , detection limit , protease inhibitor (pharmacology) , peptide , chromatography , monomer , biophysics , enzyme , biochemistry , organic chemistry , biology , molecule , physics , quantum mechanics , human immunodeficiency virus (hiv) , antiretroviral therapy , viral load , polymer , immunology
We have established a real‐time and label‐free fluorescence turn‐on strategy for protease activity detection and inhibitor screening via peptide‐induced aggregation‐caused quenching of a perylene probe. Because of electrostatic interactions and high hydrophilicity, poly‐ l ‐glutamic acid sodium salt (PGA; a negatively charged peptide) could induce aggregation of a positively charged perylene probe (probe 1) and the monomer fluorescence of probe 1 was effectively quenched. After a protease was added, PGA was enzymatically hydrolyzed into small fragments and probe 1 disaggregated. The fluorescence recovery of probe 1 was found to be proportional to the concentration of protease in the range from 0 to 1 mU/ml. The detection limit was down to 0.1 mU/ml. In the presence of a protease inhibitor, protease activity was inhibited and fluorescence recovery reduced. Moreover, we demonstrated the potential application of our method in a complex mixture sample including 1% human serum. Our method is simple, fast and cost effective.