A homogeneous assay of kinase activity that detects phosphopeptide using fluorescence polarization and zinc
| Autor: | John E. Scott, John W. Carpenter |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Phosphopeptides
Time Factors Biophysics Fluorescence Polarization Peptide Biochemistry Enzyme Inhibitors Phosphorylation Kinase activity Bovine serum albumin Protein kinase A Protein Kinase Inhibitors Molecular Biology chemistry.chemical_classification Chromatography Dose-Response Relationship Drug biology Chemistry Phosphopeptide Cell Biology Zinc Sulfate Phosphorylated Peptide Scintillation proximity assay biology.protein Oligopeptides Protein Kinases Fluorescence anisotropy |
| Zdroj: | Analytical Biochemistry. 316:82-91 |
| ISSN: | 0003-2697 |
| Popis: | Homogeneous antibody-free assays of protein kinase activity have great utility in high-throughput screening in support of drug discovery. In an effort to develop such an assay, we have used a pair of fluorescein-labeled peptides of identical amino acid sequence with and without phosphorylation on serine to mimic the substrate and product, respectively, of a kinase. Using fluorescence polarization (FP), we have demonstrated that a mixture of zinc sulfate, phosphate-buffered saline, and bovine serum albumin added to the peptides dramatically and differentially increased the fluorescence polarization of the phosphorylated peptide over its nonphosphorylated derivative. A similar FP differential was observed using different peptide pairs, though the magnitude varied. The FP values obtained using this method were directly proportional to the fraction of phosphopeptide present. Therefore, an FP assay was developed using a proprietary kinase. Using this FP method, linear reaction kinetics were obtained in enzyme titration and reaction time course experiments. The IC(50) values for a panel of inhibitors of kinase activity were determined using this FP method and a scintillation proximity assay. The IC(50) values were comparable between the two methods, suggesting that the zinc FP assay may be useful as an inexpensive high-throughput assay for identifying inhibitors of kinase activity. |
| Databáze: | OpenAIRE |
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