Institute of Chemistry, University of Tartu, Estonia.
ACS Chem Biol. 2011 Oct 21;6(10):1052-62. doi: 10.1021/cb200120v. Epub 2011 Aug 1.
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 background fluorescence of organic compounds and autofluorescence 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 μs 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.
基于时间分辨发光的分析方法在复杂生物溶液和活细胞中的测量具有很大的潜力，因为可以很容易地将测量信号与有机化合物的纳秒寿命背景荧光和细胞的自发荧光区分开来。在本研究中，我们发现含噻吩或硒吩的杂芳环部分（发光供体）与蛋白激酶（PK）的嘌呤结合口袋结合会诱导长寿命的磷光信号，该信号通过与靠近发光供体的荧光染料的有效能量转移而大大增强。与靶激酶结合时具有 19-266 μs 发光寿命的开发的 ARC-Lum 探针可用于测定碱性 PK 的活性、PK 抑制剂的表征以及 cAMP 传感器。ARC-Lum 探针还用于测定抑制剂与蛋白激酶 A（PKAc）催化亚基结合的动力学参数。通过 Forskolin 实时有效地监测 PKA 的激活，以及通过抑制剂 H89 将 ARC-Lum 探针从其与 PKA 的复合物中置换出来，在活细胞中进行了实验。所发现的现象，即芳香探针的蛋白质诱导的长寿命发光，很可能与所有 PK 和许多其他蛋白质都有关。
