A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases.

Journal article

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.

Authors: McNeill, L; Bethge, L; Hewitson, K; Schofield, C

• Publication status: Published
• Journal: Analytical biochemistry
• Volume: 336
• Issue: 1
• Pages: 125-131
• Publication date: 2005-01-01
• DOI: 10.1016/j.ab.2004.09.019
• EISSN: 1096-0309
• ISSN: 0003-2697
• Language: English
• Keywords: Repressor Proteins; Oxygenases; Transcription Factors; Spectrometry, Fluorescence; Procollagen-Proline Dioxygenase; Kinetics; Ketoglutaric Acids; Phenylenediamines