Cycling Assay for Nicotinamide Adenine Dinucleotides: NaCl Precipitation and Ethanol Solubilization of the Reduced Tetrazolium

The spectrophotometric cycling assay for the determination of nicotinamide adenine dinucleotides involving 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide as the final electron acceptor has been improved. The enzyme reaction is stopped by the addition of high concentrated NaCl, which accelerates the precipitation of the formazan. After isolation from the assay medium, the formazan is easily solubilized in ethanol. This allows more flexibility and sensitivity, offering additionally the possibility to reduce the amount of reagents required. This method was applied for extracts of green tissues of higher plants, and the results did not differ from those obtained with the original procedure.