title: Homogeneous competitive assay of ligand affinities based on quenching fluorescence of tyrosine/tryptophan residues in a protein via Főrster-resonance-energy-transfer.
abstract: A new homogeneous competitive assay of ligand affinities was proposed based on quenching the fluorescence of tryptophan/tyrosine residues in a protein via Főrster-resonance-energy-transfer using a fluorescent reference ligand as the acceptor. Under excitation around 280 nm, the fluorescence of a protein or a bound acceptor was monitored upon competitive binding against a nonfluorescent candidate ligand. Chemometrics for deriving the binding ratio of the acceptor with either fluorescence signal was discussed; the dissociation constant (K(d)) of a nonfluorescent candidate ligand was calculated from its concentration to displace 50% binding of the acceptor. N-biotinyl-N'-(1-naphthyl)-ethylenediamine (BNEDA) and N-biotinyl-N'-dansyl-ethylenediamine (BDEDA) were used as the reference ligands and acceptors to streptavidin to test this new homogeneous competitive assay. Upon binding of an acceptor to streptavidin, there were the quench of streptavidin fluorescence at 340 nm and the characteristic fluorescence at 430 nm for BNEDA or at 525 nm for BDEDA. K(d) of BNEDA and BDEDA was obtained via competitive binding against biotin. By quantifying BNEDA fluorescence, K(d) of each tested nonfluorescent biotin derivative was consistent with that by quantifying streptavidin fluorescence using BNEDA or BDEDA as the acceptor. The overall coefficients of variation were about 10%. Therefore, this homogeneous competitive assay was effective and promising to high-throughput-screening.
authors.affiliation: Unit for Biotransformation and Protein Biotechnology, Chongqing Key Laboratory of Biochemical & Molecular Pharmacology, Chongqing Medical University, Chongqing 400016, China.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Xie
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Yang
authors.forename: Jun
authors.initials: J
authors.lastname: Pu
authors.forename: Yunsheng
authors.initials: Y
authors.lastname: Zhao
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Guoming
authors.initials: G
authors.lastname: Xie
authors.forename: Jun
authors.initials: J
authors.lastname: Zheng
authors.forename: Huidong
authors.initials: H
authors.lastname: Yuan
authors.forename: Fei
authors.initials: F
authors.lastname: Liao
mesh_terms: D001667:Binding, Competitive; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays / Q000379:methods*; D008024:Ligands; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D019809:Streptavidin; D014364:Tryptophan; D014443:Tyrosine
chemical_list: D008024:Ligands; D011506:Proteins; D014443:Tyrosine; D014364:Tryptophan; D019809:Streptavidin