IMMOBILIZED-ENZYME ELECTRODE FOR NICOTINAMIDE ADENINE-DINUCLEOTIDE (REDUCED FORM) (NADH) SENSING AND APPLICATION TO THE KINETIC-STUDIES OF NADH DEPENDENT DEHYDROGENASES

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, K(m) values, and maximum velocities, V(m) values), which were in satisfactory agreement with those determined by a conventional spectrophotometric method.