A novel spectrophotometric method for the enzymatic determination of NAD+ and NADH

The theory and practice of a novel spectrophotometric method for the enzymatic determination of NAD+ and NADH is described. The method can not discriminate between NAD+ and NADH, but determines the concentration of the sum of both nucleotides. The method is based on the bleaching of p-nitroso-N,N-dimethylaniline (NMDA) (epsilon 440 nm = 35400 M-1cm-1) with NADH, in the presence of ethanol and yeast alcohol dehydrogenase, under the conditions of enzymatic cycling (ethanol > NDNA > NAD/H). The initial rates of -NDMA bleaching are proportional to the concentration of NAD+ or NADH, in a broad range from 10 nM to 100 microM.