Journal
RSC ADVANCES
Volume 11, Issue 17, Pages 10375-10380Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/d1ra01547h
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Funding
- National Research Foundation of Korea (NRF) - Korea government (MSIT) [NRF-2020R1A2B5B01002392]
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The real-time kinase assay method using a ratiometric fluorescence probe can be applied to various small-molecule kinases. It tracks the reversible interchange of ATP and ADP, facilitating monitoring of phosphorylation and dephosphorylation in small-molecule kinases. The fluorescence change can be observed with a simple light source by the naked eye, making it a convenient method for enzyme activity analysis.
A real-time kinase assay method based on a ratiometric fluorescence probe that can be applied to various small-molecule kinases is described herein. The probe can trace the reversible interchange of ATP and ADP, which is a common phenomenon in most small-molecule kinase reactions, by a ratiometric fluorescence change. This property facilitates the monitoring of phosphorylation and dephosphorylation in small-molecule kinases, whereas most of the existing methods focus on one of these reactions. To prove the applicability of this method for small-molecule kinase assays, hexokinase and creatine kinase, which phosphorylate and dephosphorylate substrates, respectively, were analyzed. The ratiometric fluorescence change was correlated with the enzyme activity, and the inhibition efficiencies of the well-known inhibitors, N-benzoyl-d-glucosamine and iodoacetamide, were also monitored. Notably, the change in fluorescence can be observed with a simple light source by the naked eye.
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