An enzymatic cycling procedure for NAD+ using an irreversible reaction with NAD+-peroxidase

✍ Scribed by C. Cox; P. Camus; J. Buret; J. Duvivier

• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 671 KB
• Volume: 119
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(82)90684-4

✦ Synopsis

A new enzymatic cycling procedure specific for NAD( H) utilizing an irreversible reaction in the cycling step and catalyzed by N AD+-peroxidase (EC 1.11.1.1) is described. The second enzyme is glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The accumulated 6-phosphogluconate is measured with 6-phosphogluconate dehydrogenase (EC 1.1.1.44) and the formed NADPH is monitored fluorimetrically. It is possible to increase the sensitivity by producing two fluorescent molecules per molecule of 6-phosphogluconate with a more sophisticated indicator reagent. The amplification is about 4000-fold/h and is sufficient to detect 25 X lo-l5 mol NAD(H) in the sample. The system is linear over a wide range of NAD(H) concentrations. The blank values are low due to the purification of the cycling enzymes by gel-filtration chromatography. This cycling system is not designed to measure NAD(H) per se but to be used in immunoassays with NAD+ as label.

' NADH