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Thiol reactive dyes as probes for kinase assays
Author(s) -
Chiku Taurai,
Pullela Phani Kumar,
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.a263
Subject(s) - förster resonance energy transfer , fluorophore , chemistry , fluorescence , fluorescein , linker , quenching (fluorescence) , kinase , dtnb , biochemistry , biophysics , acceptor , microbiology and biotechnology , glutathione , enzyme , biology , physics , quantum mechanics , computer science , condensed matter physics , operating system
Protein kinases are involved in signaling, making them favorable drug targets; hence the need to develop methods adaptable to high throughput assays and screening. We recently reported a UV Vis based kinase assay (Chiku et al, J Biomol Screen 2006 11 : –853) involving UV Vis absorbing species. The assay is based on the differential reactivity of ADP(S and ATP(S toward dithiols. We now report an efficient fluorescence version of this assay, where dithionitrobenzoic acid (DTNB) is replaced by a disulfide bridged fluorophore and the assay follows the removal of fluorescence quenching. The fluorescent probe has a cystamine linker joining a blackhole quencher and fluorescein, and has negligible background fluorescence. Removal of quenching gives greater than ten fold increase in fluorescence, and the current assay has advantages over the traditional kinase assays. A similar strategy is applied using fluorescence resonance energy transfer (FRET), and in this case kinase activity is quantitated by decrease and increase in acceptor (fluorescein) and donor (coumarin) fluorescence respectively. The FRET probes offer the possibility of in cell quantitation of kinase activity and we also pursuing a novel synthesis of ADPbS. Research supported in part by BTA grant (#LEGFY06‐12368 01KMS), AHA grant (05303072) and NIH_NSF instrumentation grants (S10RR019012 and CHE‐0521323).