Immobilized-enzyme electrode for nicotinamide adenine dinucleotide (reduced form) (NADH) sensing and application to the kinetic studies of NADH dependent dehydrogenases. |
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Authors: | H C Chang A Ueno H Yamada T Matsue I Uchida |
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Affiliation: | Department of Molecular Chemistry and Engineering, Faculty of Engineering, Tohoku University, Japan. |
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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. |
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