Silver Nanoclusters-Based Fluorescence Assay of Protein Kinase Activity and Inhibition

• Published in: Analytical chemistry (Washington) Vol. 87; no. 1; pp. 693 - 698
• Main Authors: Shen, Congcong, Xia, Xiaodong, Hu, Shengqiang, Yang, Minghui, Wang, Jianxiu
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06.01.2015
• Subjects: Adenosine diphosphate; Adenosine triphosphatase; adenosine triphosphate; Adenosine Triphosphate - metabolism; Assaying; ATP; Biological Assay; cAMP-dependent protein kinase; Cyclic AMP-Dependent Protein Kinases - chemistry; Cyclic AMP-Dependent Protein Kinases - metabolism; Enzyme Inhibitors - pharmacology; Fluorescence; HeLa Cells; Humans; hydrolysis; Inhibition; Inhibitors; Kinases; Metal Nanoparticles - chemistry; monitoring; Monitoring methods; nanoparticles; Nanostructure; Nanostructures - chemistry; oligonucleotides; Peptides; Phosphorylation; Silver; Silver - chemistry; Spectrometry, Fluorescence
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/ac503492k

A simple and sensitive fluorescence method for monitoring the activity and inhibition of protein kinase (PKA) has been developed using polycytosine oligonucleotide (dC12)-templated silver nanoclusters (Ag NCs). Adenosine-5′-triphosphate (ATP) was found to enhance the fluorescence of Ag NCs, while the hydrolysis of ATP to adenosine diphosphate (ADP) by PKA decreased the fluorescence of Ag NCs. Compared to the existing methods for kinase activity assay, the developed method does not involve phosphorylation of the substrate peptides, which significantly simplifies the detection procedures. The method exhibits high sensitivity, good selectivity, and wide linear range toward PKA detection. The inhibition effect of kinase inhibitor H-89 on the activity of PKA was also studied. The sensing protocol was also applied to the assay of drug-stimulated activation of PKA in HeLa cell lysates.