Thiol reactive probes: Applications in kinase assays and thiol detection
A general kinase reaction was developed based on the differential reactivity of ATPβS and ADPβS torward bis -dithio nitrobenzoic acid (DTNB). ADPβS, an analog of ADP, the common product of all kinase (or ATPase) reactions is detected after undergoing a disulfide exchange reaction with DTNB to yield the intensely yellow-colored thionitrobenzoate (TNB) anion. The fluorescence version of the kinase assay utilizing dithio-bridged homo-dye dimers has also been tested and is adversely affected by high background fluorescence due to insufficient internal dye quenching. A marked reduction in the background fluorescence was achieved by the use of (FRET-matched) dithio-bridged hetero-dye dimers. A Z' factor of 0.6 was obtained using a fluorescein and rhodamine dithio-bridged dye. Kinetic data of the kinase reaction using hexokinase and glucose suggest tighter binding of ATPβS compared to ATP. Likewise, glutathione (GSH), the most abundant cellular thiol, can be detected by a disulfide exchange reaction with dithio-bridged hetero-dye dimers leading to a change in the fluorescence properties of the dye core(s). 1 mM to 10 mM changes in GSH, corresponding to the physiologically relevant GSH levels, showed an almost linear response to net change in fluorescence of the dyes. The cellular application of the dyes was demonstrated through the use of wild-type E. Coli cells and protein reductase deficient cells, in which higher thiol levels were observed in the wild-type cell line. Exposure of zebrafish embryos by micro injection and indirect exposure resulted in the localization of fluorescence in the chorion layer.
"Thiol reactive probes: Applications in kinase assays and thiol detection"
(January 1, 2008).
Dissertations (1962 - 2010) Access via Proquest Digital Dissertations.