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[摘要]：Time-resolved luminometry-based assays have great potential for measurements in complicated biological solutions and living cells as,the measured signal can be easily distinguished from nanosecond lifetime back ground fluorescence of organic compounds and autofluotescence of cells. In the present,study we discovered that binding of a thiophene- or a selenophene-containing heteroaromatic moiety (luminescence donor) to the purine-binding pocket of a protein kinase (PK) induces long lifetime photoluminescence signal that is largely intensified through efficient energy transfer to a fluorescent dye present in close proximity to the luminescence donor. The developed ARC-Lum probes possessing 19-266 mu s luminescence lifetime when associated with the target kinase can be used for determination of activity of basophilic PKs, characterization of inhibitors of PKs, and as cAMP sensors. An ARC-Lum probe was also used for the determination of kinetic parameters of inhibitor binding to the catalytic subunit of protein kinase A (PKAc). Effective real-time monitoring of the activation of PKA by Forskolin and the displacement of an ARC-Lum probe from its complex with PKA by inhibitor H89 was performed in live cells. The discovered phenomenon, protein-induced long lifetime luminescence of aromatic probes is very likely to occur with all PKs and many other proteins. |
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