Electron-transfer function of NAD+-immobilized alginic acid

Polymerized NAD+ (Alg-NAD+) was prepared and its electrochemical properties were investigated. NAD+ has been covalently immobilized at the carboxyl group of alginic acid using water soluble carbodiimide (EDC) and then Alg-NAD+s of various NAD+ density were obtainable depending on NAD+ concentration in the reaction mixture. Absorbance of 260 nm of Alg-NAD+s showed that 3.4 to 17.6% of carboxyl groups of alginic acid were coupled with NAD+. The coenzyme activity of immobilized NAD+ has reached 80 to 90% on each Alg-NAD+. A cathodic peak in the cyclic voltammogram of Alg-NAD+ appeared at -1.2 V (vs. SCE) corresponding to the reduction wave of free NAD+. The anodic wave of NAD dimer was not observed in the presence of 2.0 mM methyl viologen and 5 units of diaphorase and NAD+ immobilized on the composite electrode could be reduced to the normal NADH. The ratio of apparent diffusion coefficient (Dapp.) of Alg-NAD+ and free NAD+ was evaluated from the variation of ipc with the square root of sweep rate (v 1/2). Despite the high molecular weight of Alg-NAD+, Dapp. Alg-NAD+/Dapp. free NAD+ are larger than that expected. These results indicate that electron transfer occurred effectively between each NAD+ molecule immobilized onto the polymer chain. It is also confirmed by a conjugated redox enzyme reaction with Alg-NAD+.