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 a n enzyme reactor containing coimmobilized glycerol dehydrogenase (GDH) and diaphorase. The product from the diaphorase catalysis, hexacyanof errate(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 s ignal increased another 88 times, resulting in a signal amplification of 13 300 times. Equations are derived for the amplification factor an d 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 filtrat ion column and the purified enzyme showed a specificity toward NAD(+), compared to NADP(+), that was higher than 99.9%. Due to the NAD(+) sp ecificity of the purified GDH, the enzyme amplification system reporte d 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 magn itude better than that of alcohol dehydrogenase, which is the enzyme c ommonly used for NAD(+)-specific detection in these applications.