Application of the Enzymic Electric Cell Method to the Activity Assay of NAD-linked Dehydrogenases

Abstract

An enzymic electric cell was constructed with a saturated calomel electrode (cathode) and an enzymic electrode (anode) which consisted of a glassy carbon electrode and a mixture containing an NAD-linked dehydrogenase, NAD⁺, N-methylphenazonium methosulfate and a substrate, and the short-circuit current of the cell was measured. NAD-linked dehydrogenases tested were lactate dehydrogenase [EC 1. 1. 1. 27], alcohol dehydrogenase [EC 1. 1. 1. 1], and malate dehydrogenase [EC 1. 1. 1. 37]. In this cell, electrons from the substrate in the anode mixture were enzymatically transferred to NAD⁺, and were then transferred to the glassy carbon electrode via N-methyl-phenazonium methosulfate as an intermediary electron carrier, eventually being observed as the short-circuit current of the cell. As long as the enzymic step limited the overall rate of electron transfer, the current was proportional to the amount of the enzyme in the anode container. Thus, the activities of NAD-linked dehydrogenases could be assayed by this “enzymic electric cell method” in the same way as hydrogenase [EC 1. 12. 2. 1] (T. Yagi et al. (1975) J. Biochem. 78, 443-454).