A sensitive fluorometric assay is reported for label-free detection of the activity of protein kinase (PKA) and of its inhibitors. It is based on the europium(III)-modulated fluorescence of peptide-stabilized gold nanoclusters (peptide-AuNCs). Both adenosine-5′-triphosphate (ATP) and adenosine-5′-diphosphate (ADP) (formed from ATP by PKA-catalyzed hydrolysis) enhance the fluorescence of peptide-AuNCs with its excitation/emission peaks of 330/405 nm. The addition of Eu(III) quenches the fluorescence of the ATP/peptide-AuNC system to the original fluorescence level of peptide-AuNCs, while addition of Eu(III) has no effect on the fluorescence of the ATP/PKA/peptide-AuNC system. Based on this “turn-on” method, the activity of PKA can be detected with high sensitivity in 0.05–1.6 U⋅mL−1 activity range, with a detection limit of 0.02 U⋅mL−1 (3σ). The feasibility of this method for screening of inhibitors was also studied. The IC50 value for the commercially available inhibitor H-89 was found to be 43 nM. Moreover, the detection scheme was also applied to monitoring the drug-stimulated activation of PKA in HepG-2 cell lysates.
Microchimica Acta – Springer Journals
Published: Jun 14, 2017
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