A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases.
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.
McNeill
L A
LA
Chemistry Research Laboratory, The Department of Chemistry and the Oxford Centre for Molecular Sciences, Mansfield Road, Oxford OX1 3TA, UK.
Bethge
L
L
Hewitson
K S
KS
Schofield
C J
CJ
D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D006899:Mixed Function Oxygenases; D010105:Oxygenases / Q000032:analysis*; D010655:Phenylenediamines / Q000737:chemistry; D011393:Procollagen-Proline Dioxygenase / Q000032:analysis; D012097:Repressor Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000378:metabolism
D007656:Ketoglutaric Acids; D010655:Phenylenediamines; D012097:Repressor Proteins; D014157:Transcription Factors; C034193:1,2-diaminobenzene; D006899:Mixed Function Oxygenases; D010105:Oxygenases; C439790:HIF1AN protein, human; D011393:Procollagen-Proline Dioxygenase