Date of Award

Fall 2001

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Daniel Sem

Second Advisor

Mark Steinmetz

Third Advisor

Scott Reid

Abstract

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. We also report an improved version of our previously reported fluorescent-dithio probes for detecting cellular thiols, referred to as PMR-CYS-FITC, where PMR is p-methyl red (aka dabcyl), a fluorescein quencher. Unlike most thiol-reactive probes, PMR-CYS-FITC can also detect certain disulfides, making it useful for `disulfide proteomics'. PMR-CYS-FITC was used to identify new disulfide-containing protein(s) in the zebrafish chorion, with potential role(s) in protecting the embryo during development. Confocal microscopy of the embryo, followed by in-gel staining and imaging of proteins, then tandem MS, led to our discovery of two novel disulfide-containing proteins in the fish chorion, lipovitellin and C-reactive protein (CRP). CRP is a well-known biomarker for infection or inflammation (associated with oxidative stress) in humans, so discovery of CRP in the zebrafish has relevance for its use as a model organism. In vitro biophysical characterization of this probe provides further explanations for the differential reactivity of disulfides, and enhanced fluorescence for some fluorescein-protein mixed disulfides.

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