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α‐Ketoglutarate Assay Based on Fluorescence Quenching by NADH
Author(s) -
Sharma Ashutosh,
Quantrill Nigel S. M.
Publication year - 1996
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp960027f
Subject(s) - thionine , fluorophore , substrate (aquarium) , cofactor , quenching (fluorescence) , chemistry , fluorescence , yield (engineering) , population , dehydrogenase , enzyme , photochemistry , chromatography , biochemistry , analytical chemistry (journal) , biology , materials science , optics , ecology , physics , demography , electrode , sociology , metallurgy , electrochemistry
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, K t . The analysis of data obtained over a range of 0−500 μM results in a constant of 2748 M − 1 . The applicability of the proposed method is demonstrated by performing the assay for α‐ketoglutarate in human urine. The ketoglutarate SIQ assay was not affected by the background interference that is inherent to this complex matrix.