title: Selective determination of cysteines through precolumn double-labeling and liquid chromatography followed by detection of intramolecular FRET.
abstract: In this paper we introduce a novel approach for highly selective and sensitive analysis of cysteines (glutathione, cysteine, and homocysteine). This method is based on the detection of intramolecular fluorescence resonance energy transfer (FRET) in a liquid chromatography (LC) system after double-labeling of the amino and sulfhydryl groups of the cysteines. In this detection process, we monitored the FRET between the amine-derivatized and thiol-derivatized fluorophores. We screened 16 combinations of fluorescent reagents. As a result, FRET occurred most effectively when the sulfhydryl and amino groups of the cysteines were derivatized with 7-diethylamino-3-[{4'-(iodoacetyl)amino}phenyl]-4-methylcoumarin (DCIA, Ex/Em 390/480 nm) and 4-fluoro-7-nitrobenz-2-oxo-1,3-diazole (NBD-F, Ex/Em 480/540 nm), respectively, in this order. The double-labeled cysteines emitted NBD-F fluorescence (540 nm) through an intramolecular FRET process when they were excited at the wavelength of maximum excitation of DCIA (390 nm). The generation of FRET was confirmed by comparison with analysis of n-amylamine or tryptophan (amines without a sulfhydryl group) and 6-mercaptohexanol (thiol without an amino group) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the double-labeled cysteines (DCIA and NBD-F) when performing LC on an ODS column with isocratic elution. The limits of quantification (signal-to-noise ratio = 10) and detection (signal-to-noise ratio = 3) for the cysteines, for a 20-μL injection volume, were in the range 150-670 fmol and 46-200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of a system which takes advantage of conventional detection of the derivatives. Furthermore, this method provides sufficient selectivity and sensitivity to determine the total cysteines present in the plasma of healthy humans.
authors.affiliation: Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Johnan, Fukuoka 814-0180, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Yoshitake
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nohta
authors.forename: Naoko
authors.initials: N
authors.lastname: Sejima
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Todoroki
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Yoshida
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Yamaguchi
mesh_terms: D002853:Chromatography, Liquid; D003545:Cysteine / Q000097:blood*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D012016:Reference Values; D012680:Sensitivity and Specificity; D013194:Staining and Labeling
chemical_list: D003545:Cysteine