[15] Determination, purification, and characterization of α-NADH and α-NADPH

This chapter describes the two methods for the quantitative determination of small amounts of α-anomers of nicotinamide adenine dinucleotide (NAD), NADH, nicotinamide adenine dinucleotide phosphate (NADP), and NADPH in the presence of large amounts of β-anomers. These methods use ion-exchange chromatography or reversed-phase chromatography combined with enzymatic oxidation of β-NADH and β-NADPH by lactate dehydrogenase (LDH) and glutathione reductase, respectively. They are used to determine the rate and equilibrium constants of the α–β anomerization reactions of NADH and NADPH. Because commercially available α-NADH preparations are not pure, a method for the preparation of 20- to 50-mg amounts of α-NADH with purity better than 96% is presented and spectrophotometric and fluorometric properties of these preparations are investigated. The spontaneous α-to-β and β-to-α anomerization reaction of NADH is investigated quantitatively by following the α-to-β reaction either by measuring the residual absorption or by using the relatively high specificity of dehydrogenases for β-NADH, directly measuring anomerization velocity in the presence of large amounts of dehydrogenases.