Enzymatic amplification for spectrophotometric and electrochemical assays of NAD+ and NADH.
Five different procedures are presented for the enzymatic assay of the sum of NAD+ and NADH concentrations. They are based on the principle of amplification by cycling. The reactions involve oxidation of the formate ion, ethanol, glucose, or carnitine catalyzed by the corresponding dehydrogenases. The detection reactions are based on the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT)/INT-formazan and ferricyanide/ferrocyanide couples and use a diaphorase. Two of the systems presented--with formate ion and ethanol--were coupled with spectrophotometric detection. The absorbance measurement values were multiplied by 3 in the first case and by 20 in the second, with respect to the values that would have been obtained in the same conditions without the amplification system. The accessible concentration ranges were between 0.05 and 100 microM approximately. Three systems--with formate ion, carnitine, and glucose--used an electrochemical detection based on oxidation of the ferrocyanide ion. The response times were of the order of 10 min and the precision of about 5%. The first brought to light some difficulties concerning the design of such devices. For the second, the proportionality constant had a value of the order of 0.25 microA.microM-1 and an accessible concentration range between 0.2 and 40 microM. The third allowed more precise assays for lower concentration values: between 0.02 and 1.5 microM, with a proportionality constant of 0.49 microA.microM-1. Emphasis was placed on the adaptation possibilities of these systems as a function of the assay requirements.
Laboratoire de Génie Chimique, URA CNRS 192, Université Paul Sabatier, Toulouse, France.
A
A
Bergel
J
J
Souppe
M
M
Comtat
D004563:Electrochemistry; D004798:Enzymes; D000431:Ethanol / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D009243:NAD / Q000032:analysis*; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet
D004798:Enzymes; D009243:NAD; D000431:Ethanol; D005947:Glucose