Electrolytic regeneration of the reduced from the oxidized form of immobilized NAD.

A covalently bound adduct of nicotinamide adenine dinucleotide (NAD) with alginic acid has been found to be enzymatically active and to undergo electrochemical oxidation or reduction without significant loss of its enzymatic activity. The preparation of the adduct itself (from NAD+, alginic acid, and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate) is also accomplished with substantially complete retention of enzymatic activity. This adduct has been converted from the oxidized to the reduced form by controlled potential electrolysis using mercury and stainless-steel electrodes. This electrolytically produced NADH complex could be oxidized again to the enzymatically active NAD+ complex by enzymatic reaction with the proton acceptor, 2,6-dichlorophenol indophenol, as catalyzed by diaphorase. Using this electrolytic method with immobilized NAD, it is now possible to carry out redox reactions in which NADH is enzymatically oxidized to NAD+, with the simultaneous electrolytic regeneration of the reduced form, NADH, from the oxidized form, NAD+, produced in the enzymatic reaction.