Selective enzyme amplification of NAD+/NADH using coimmobilized glycerol dehydrogenase and diaphorase with amperometric detection
✍ Scribed by Karin Kronkvist; Katarina Wallentin; Gillis Johansson
- Publisher
- Elsevier Science
- Year
- 1994
- Tongue
- English
- Weight
- 731 KB
- Volume
- 290
- Category
- Article
- ISSN
- 0003-2670
No coin nor oath required. For personal study only.
✦ Synopsis
A flow system for substrate recycling of NAD+/NADH was set up with an enzyme reactor containing coimmobiliied glycerol dehydrogenase (GDH) and diaphorase. The product from the diaphorase catalysis, hexacyanoferrate(II), was detected amperometrically at a glassy carbon electrode. The amplification factor was 150 for a reactor volume of 100 ~1 at a flow-rate of 0.5 ml/min. With a stopped flow of four minutes, the signal increased another 88 times, resulting in a signal amplification of 13 300 times. Equations are derived for the amplification factor and used for a discussion of the optimization of amplification systems. The K, for GDH with glycerol as a substrate was found to be 5 x 10m3 M at pH 8.0. GDH from Celhlomonus sp. was purified on a gel filtration column and the purified enzyme showed a specificity toward NAD+, compared to NADP+, that was higher than 99.9%. Due to the NAD+ specificity of the purified GDH, the enzyme amplification system reported here could be used in detection systems for enzyme immunoassays when using alkaline phosphatase as a label and NADP+ as a substrate. The stability of immobilized GDH and diaphorase is several orders of magnitude better than that of alcohol dehydrogenase, which is the enzyme commonly used for NAD+-specific detection in these applications.