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Peptide Biosensors for the Electrochemical Measurement of Protein Kinase Activity
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Department of Chemistry, Faculty of Science.
, ‡Department of Biochemistry, Schulich School of Medicine and Dentistry.
Section:Abstract
The kinase activities are elucidated using the novel redox-active cosubstrate adenosine 5′-[γ-ferrocene] triphosphate (Fc-ATP), which enables the kinase-catalyzed transfer of a redox active γ-phosphate-Fc to a hydroxyamino acid. In this report, a versatile electrochemical biosensor is developed for monitoring the activity and inhibition of a serine/threonine kinase, casein kinase 2 (CK2), and protein tyrosine kinases, Abl1-T315I and HER2, in buffered solutions and in cell lysates. The method is based on the labeling of a specific phosphorylation event with Fc, followed by electrochemical detection. The electrochemical response obtained from the “ferrocenylated” peptides enables monitoring the activity of the kinase and its substrate, as well as the inhibition of small molecule inhibitors on protein phosphorylation. Kinetic information was extracted from the electrochemical measurements for the determination of Km and Vm values, which were in agreement with those previously reported. Kinase reactions were also performed in the presence of well-defined inhibitors of CK2, 4,5,6,7-tetrabromo-2-azabenzimidazole, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, and E-3-(2,3,4,5-tetrabromophenyl)acrylic acid as well as the nonspecific kinase inhibitors, staurosporine and N-benzoylstaurosporine. On the basis of the dependency of the Fc signal on inhibitor concentration, Ki of the inhibitors was estimated, which were also in agreement with the literature values. The performance of the biosensor was optimized including the kinase reaction, incubation with Fc-ATP, and the small molecule inhibitors. Peptide modified electrochemical biosensors are promising candidates for cost-effective in vitro kinase activity and inhibitor screening assays.
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This article has been cited by 8 ACS Journal articles (5 most recent appear below).
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Label-Free Fluorescent Detection of Protein Kinase Activity Based on the Aggregation Behavior of Unmodified Quantum Dots
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Xiahong Xu, Xin Liu, Zhou Nie, Yuliang Pan, Manli Guo, and Shouzhuo YaoAnalytical Chemistry2011 83 (1), 52-59Herein, we present a novel label-free fluorescent assay for monitoring the activity and inhibition of protein kinases based on the aggregation behavior of unmodified CdTe quantum dots (QDs). In this assay, cationic substrate peptides induce the selective ...
Highly Sensitive Electrogenerated Chemiluminescence Biosensor in Profiling Protein Kinase Activity and Inhibition Using Gold Nanoparticle as Signal Transduction Probes
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History
- Published In Issue December 15, 2008
- Article ASAPNovember 07, 2008
- Received: June 13, 2008
Accepted: October 14, 2008
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