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Fluorescent probes for in vivo redox potential measurement and thiol rich protein function
Author(s) -
Pullela Phani Kumar,
Chiku Taurai,
Carvan Michael J,
Sem Daniel S
Publication year - 2006
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.20.4.a74-a
Subject(s) - intracellular , cysteine , glutathione , thiol , chemistry , redox , biochemistry , biophysics , zebrafish , microbiology and biotechnology , biology , enzyme , organic chemistry , gene
Fluorescence study of thiol rich proteins like metalothioneins and intra‐cellular thiols have met with poor success. We synthesized a set of DSSA probes to study intracellular thiols and surface rich proteins. These reagents are shown to cross bacterial cell wall and are monitored by increase in fluorescence due to reaction of GSH with DSSA probe. When uptake studies are done with E.coli Origami cells, which are deficient in glutathione synthesis, much less fluorescence was observed. This suggests the utility of the DSSA probes for studying changes in intracellular GSH and changes in redox potential. The DSSA probes have redox potentials of around – 0.6 V, suggesting that their use in vivo will not disrupt intracellular redox state by oxidizing the cell. As a complement to these bacterial uptake studies, the DSSA probes are used to monitor thiol levels in a eukaryotic system: developing zebrafish embryos. Embryos were either incubated with the probe, or probe was microinjected past the chorion layer. In both cases, significant accumulation of probe was observed in the chorion suggesting the presence of thiol‐rich proteins. We propose that, zebrafish have a mechanism by which the surface cysteine rich proteins serve as protective layer. Considering these results, these DSSA probes should have utility for monitoring thiol rich protein function, dynamics and importantly, measuring redox potentials of intracellular components.