A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases
| Autor: | Luke A. McNeill, Christopher J. Schofield, Kirsty S. Hewitson, L. Bethge |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
chemistry.chemical_classification
Oxygenase Biophysics Procollagen-Proline Dioxygenase Cell Biology Phenylenediamines Biochemistry Fluorescence Mixed Function Oxygenases Repressor Proteins chemistry.chemical_compound Kinetics Enzyme Spectrometry Fluorescence chemistry 2-Oxoglutarate Oxygenases Ketoglutaric Acids Derivatization Molecular Biology Transcription Factors |
| Zdroj: | Analytical biochemistry. 336(1) |
| ISSN: | 0003-2697 |
| Popis: | A widely used generic assay for 2-oxoglutarate-dependent oxygenases relies upon monitoring the release of 14CO2 from labeled [1-14C]-2-oxoglutarate. We report an alternative assay in which depletion of 2-oxoglutarate is monitored by its postincubation derivatization with o-phenylenediamine to form a product amenable to fluorescence analysis. The utility of the procedure is demonstrated by assays with hypoxia-inducible factor hydroxylases where it was shown to give results similar to those reported with the radioactive assay, but it is more efficient and readily adapted to a multiwell format. The process should be amenable to the assay of other 2-oxoglutarate-consuming enzymes and to the discovery of inhibitors. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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