RECYCLING ANALYSIS OF NICOTINAMIDE-ADENINE DINUCLEOTIDE PHOSPHATES (NADP AND NADPH)

A simple and sensitive analysis of NADP and NADPH is described. It uses the glucose-6-phosphate dehydrogenase-glutathione reductase system with detection of reduced glutathione through reaction with 5,5′-dithiobis(2-nitrobenzoic acid), Ellmans reagent. The rate of reaction is constant for several hours, and the analytical system does not react with NAD or NADH. Linear standard curves are obtained with 1.25 μg of each enzyme in 1 ml assay mixture, while higher amounts of enzyme in most cases cause nonlinear standard curves. The dynamic range is from approximately 0.2 to 400 pmol corresponding to 0.8 nm to 1.6 μm in the extracts. The analytical reactions are inhibited by salts, but reliable quantitation is obtained through the use of internal standards. The applicability to acid and alkaline extracts is investigated, and the analysis is compared with a fluorometric endpoint method. The two methods of analysis give the same analytical results. © 1979 Academic Press, Inc.