NADP⁺- Based Enzyme Assay for D-isocitrate

Abstract

Abstract A novel procedure for the measurement of D-isocitrate is described that utilizes the fluorescence quenching of thionine by the reduced form of the cofactor NADPH, which results from the isocitrate dehydrogenase-catalyzed conversion of D-isocitrate to 2-oxoglutarate. The procedure is termed substrate induced-quenching (SIQ) assay and results in a fluorescence-quenching constant (KSIQ) of 4443 M−1 obtained from data collected over the range of 0–500 μM isocitrate.