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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.

A novel optical procedure for measuring L-glutamate concentrations is described that exploits the quenching of thionine fluorescence by NADH, the latter being generated by the reduction of NAD in an enzymatic reaction between Lglutamate dehydrogenase (GIDH) and Lglutainate. A substrate induced quenching (SIQ) constant of 660 (±140) M1 at 200 s is obtained. The reported method was investigated over the Lglutamate concentration range of 0400 jiM and offers a detection limit of6 pM.

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A novel process called substrate induced quenching (SIQ), mathematical derivation of an empirical relation describing it and based on this, a model for a dehydrogenase enzymatic system are described.. The validity of the model is shown by comparison with experimental results for the SIQ based ethanol assay involving the alcohol dehydrogenase enzyme. Various applications ofthis novel technique are discussed.