SELECTIVE ENZYME AMPLIFICATION OF NAD+/NADH USING COIMMOBILIZED GLYCEROL DEHYDROGENASE AND DIAPHORASE WITH AMPEROMETRIC DETECTION

A flow system for substrate recycling of NAD(+)/NADH was set up with an enzyme reactor containing coimmobilized 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 mu l 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-M for GDH with glycerol as a substrate was found to be 5 x 10(-3) M at pH 8.0. GDH from Cellulomonas 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.