title: Immobilized-enzyme electrode for nicotinamide adenine dinucleotide (reduced form) (NADH) sensing and application to the kinetic studies of NADH dependent dehydrogenases.
abstract: Amperometric determination of nicotinamide adenine dinucleotide (reduced form) (NADH) at an immobilized-diaphorase (Dp) electrode is described. The measurement was conducted using ferrocenylmethanol as a mediator in a stirred solution at 0.20 V versus a saturated calomel electrode. A linear relationship between the steady-state current and the concentration of NADH was found over the range 0.005-0.125 mmol dm-3. The immobilized-Dp electrode showed outstanding stability and the current response reached a steady state within 2-3 seconds upon addition of NADH. The proposed electrode was used to follow the reactions of pig heart lactate dehydrogenase and horse liver alcohol dehydrogenase. The kinetic investigation using the immobilized-Dp electrode gave the kinetic parameters (Michaelis constants, Km values, and maximum velocities, Vm values), which were in satisfactory agreement with those determined by a conventional spectrophotometric method.
authors.affiliation: Department of Molecular Chemistry and Engineering, Faculty of Engineering, Tohoku University, Japan.
authors.forename: H C
authors.initials: HC
authors.lastname: Chang
authors.forename: A
authors.initials: A
authors.lastname: Ueno
authors.forename: H
authors.initials: H
authors.lastname: Yamada
authors.forename: T
authors.initials: T
authors.lastname: Matsue
authors.forename: I
authors.initials: I
authors.lastname: Uchida
mesh_terms: D004566:Electrodes; D004800:Enzymes, Immobilized; D007700:Kinetics; D009243:NAD / Q000032:analysis*; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D009243:NAD; D009247:NADH, NADPH Oxidoreductases