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Coumarin‐Fluorescein – Based FRET Probe for Real Time Quantitation of Thiols Redox State
Author(s) -
jonhera timothy,
Pullela Phani kumar,
Sem daniel
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1059.3
Subject(s) - chemistry , fluorescein , fluorescence , förster resonance energy transfer , glutathione , redox , acceptor , thiol , bodipy , photochemistry , coumarin , titration , biophysics , biochemistry , inorganic chemistry , organic chemistry , physics , quantum mechanics , biology , enzyme , condensed matter physics
The total concentration of cellular thiols is about 30 mM, and use of current fluorescent chemical probes to quantitate these thiols inside cells is impractical. We recently reported fluorescent dithio probes (donor‐S‐S‐acceptor, DSSA) with unusually low reduction potential (− 0.6 V) that can detect changes in intracellular thiols and cross membranes of bacterial and mammalian cells, but they could not quantify thiol redox state. Here we report a new variant of the DSSA series, which has hydroxy coumarin (Ex: 320 nm, Em: 460 nm) as donor and fluorescein (Ex: 485 nm, Em: 520 nm) as acceptor. The probe was characterized in vitro by titration with glutathione in a physiologically relevant range and monitoring changes in fluorescence emission from both Coumarin and Fluorescein, upon reduction of the disulfide bond that joins this donor‐acceptor FRET pair. The ratio of fluorescence emission band intensity, changes upon reduction with glutathione, and because of the probe's low reduction potential, permits quantitative measurement of glutathione levels in the 1 – 10 mM range. This is the first demonstration of ratiometric (FRET‐based) thiol level measurement and will be enabling for quantitative assessment of thiol redox state (GSG/GSSG) inside cells. Preliminary studies in E. Coli are also presented. This research was supported by a Biomedical Technology Alliance grant (01‐KMS LEG FY06‐12368) to D.S.S