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Determination of protein kinase A activity and inhibition by using hydroxyapatite nanoparticles as a fluorescent probe

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Abstract

The authors describe a fluorometric method for the determination of the activity and inhibition of protein kinase A (PKA). In the presence of ATP, PKA catalyzes the transfer of phosphate groups from ATP to a peptide, and the generated phosphorylated peptide quenches the fluorescence (measured at excitation/emission peaks of 340/440 nm) of the hydroxyapatite nanoparticles (HAP-NPs). A linear logarithmic relationship of PKA concentrations with fluorescence intensity in the range from 1 to 50 U·L−1 was obtained, and the lower limit of detection (LOD) is 0.5 U·L−1. This is much lower than LODs reported in the literature. The PKA inhibitor H-89 was studied, and the inhibition plot has a sigmoidal shape with a half-maximal inhibitory concentration of around 750 nM of H-89. At a 4.5 nM level of H-89, fluorescence of HAP-NPs fell to levels of no PKA controls, demonstrating that the assay is a viable tool to screen for kinase inhibitors. An assay with Hela cell lysates in combination with forskolin (an activator of adenylyl cyclase) and IBMX (a phosphodiesterase inhibitor used to activate the cellular activity of PKA) resulted in decreased fluorescence of HAP-NPs. This suggests that the assay can be applied for testing in vitro cell kinase activity. In our perception, this method will enable high-throughput screening for kinase-related drugs and fluorometric enzymatic detection in various areas.

Fluorescence assay based on hydroxyapatite nanoparticles (HAP) fluorescence quenching was developed for analysis of the activity and inhibition of protein kinase A (PKA).

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Fig. 1
Fig. 2: HAP-NPs fluorescence quenching by phosphopeptide.
Fig. 3: HAP-NP based detection of PKA activity.
Fig. 4
Fig. 5
Fig. 6: HAP-NP based biosensor to screen PKA inhibitors.
Fig. 7: HAP-NP-based biosensor for measuring PKA activity in cell lysates.

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Acknowledgments

The authors thank the support of this work by the National Natural Science Foundation of China (No. 21575165) and the Hunan Provincial Science and Technology Plan Project, China (No. 2016TP1007).

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Author notes

  1. Kaina Zhang and Ke Zeng contributed equally to this work.

Authors and Affiliations

  1. College of Chemistry and Chemical Engineering, Hunan Provincial Key Laboratory of Efficient and Clean Utilization of Manganese Resources, Central South University, Changsha, 410083, China

    Kaina Zhang, Ke Zeng, Congcong Shen, Shiyu Tian & Minghui Yang

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  1. Kaina Zhang
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  2. Ke Zeng
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  3. Congcong Shen
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  4. Shiyu Tian
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  5. Minghui Yang
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Correspondence to Minghui Yang.

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Zhang, K., Zeng, K., Shen, C. et al. Determination of protein kinase A activity and inhibition by using hydroxyapatite nanoparticles as a fluorescent probe. Microchim Acta 185, 225 (2018). https://doi.org/10.1007/s00604-018-2754-1

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  • DOI: https://doi.org/10.1007/s00604-018-2754-1

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