alpha-Ketoglutarate assay based on fluorescence quenching by NADH.
A new assay procedure for the measurement of ketoglutarate concentrations is described which is based on substrate-induced quenching (SIQ) of a fluorophore. The method makes use of the photoreaction between a fluorophore (thionine) and NADH. The latter is consumed during an enzymatic reaction between ketoglutarate and L-glutamic dehydrogenase. The conversion yield of cofactor from its reduced form to oxidized forms represented as an overall change in the population of the excited state population of the fluorophore thionine. An empirical relation is described that correlates initial substrate concentration to the observed yield of the cofactor conversion via a fluorescence recovery constant,Kt. The analysis of data obtained over a range of 0-500 microM results in a constant of 2748 M-1. The applicability of the proposed method is demonstrated by performing the assay for alpha-ketoglutarate in human urine. The ketoglutarate SIQ assay was not affected by the background interference that is inherent to this complex matrix.
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Cranfield Molecular Measurement Technology Centre, Cranfield University, Bedfordshire, United Kingdom.
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D002021:Buffers; D005454:Fluorescence Polarization; D006801:Humans; D007656:Ketoglutaric Acids / Q000032:analysis* / Q000652:urine; D009243:NAD / Q000737:chemistry*; D010640:Phenothiazines / Q000737:chemistry; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014867:Water / Q000737:chemistry
D002021:Buffers; D007656:Ketoglutaric Acids; D010640:Phenothiazines; D012996:Solutions; D014867:Water; D009243:NAD; C009469:thionine