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.
Leskovac
V
V
Faculty of Technology, Bulevar Cara Lazara 1, Yugoslavia.
Trivić
S
S
Anderson
B N
BN
D000426:Alcohol Dehydrogenase / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D009243:NAD / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D009603:Nitroso Compounds; D013056:Spectrophotometry, Ultraviolet
D007202:Indicators and Reagents; D009603:Nitroso Compounds; D009243:NAD; C008651:4-nitrosodimethylaniline; D000426:Alcohol Dehydrogenase