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Fluorescent probes for thiol quantitation inside cells
Author(s) -
Pullela Phani kumar,
Chiku Taurai,
Sem Daniel S
Publication year - 2007
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.21.5.a627-a
Subject(s) - förster resonance energy transfer , glutathione , chemistry , thiol , cysteine , fluorescence , reagent , quenching (fluorescence) , intracellular , oxidative stress , acceptor , biophysics , membrane , biochemistry , organic chemistry , enzyme , biology , physics , quantum mechanics , condensed matter physics
Thiol containing compounds like glutathione and cysteine are essential for proper function of a cell. The levels of thiols determine the redox‐potential, which is associated with oxidative stress, and disease state of a cell. We recently demonstrated that DSSA (donor‐S‐S‐acceptor) dithio probes with unusually low reduction potential (− 0.6 V) are sensitive to intracellular thiols and cross the cell membranes of different bacterial and human cells (Pullela et al Anal. Biochem . 352, ( 2006 ) 265–273). Here we report fluorescence resonance energy transfer (FRET) and quenching based reagents for quantitation of thiols, with aromatic and aliphatic disulphide linkers. A possible FRET effect and up to five fold change in fluorescence intensity was observed during reaction of thiols with these reagents. Surprisingly, we observed a change in absorbance spectrum during reduction due to dye to dye quenching similar to DSSA probes. These reagents could have useful applications in biochemistry to a) quantitate changes in cellular thiol levels and glutathione during stress; b) measure effects of small molecules on intracellular thiol levels. This research is supported in part by a Biomedical Technology Alliance grant (#LEGFY06‐12368 01‐KMS) and NIH‐NSF instrumentation grants (S10RR019012 and CHE‐0521323).