Determination, Purification, and Characterization of α-NADH

Abstract

α-NADH can be determined in the presence of high concentrations of β-NADH using anion exchange HPLC combined with oxidation of β-NADH by lactate dehydrogenase. The method is suited for the detection of a large number of impurities. Residual absorption is a poor measure of α-NADH. By combination of preparative anion exchange chromatography and reversed-phase HPLC, α-NADH can be prepared with 95-98% purity starting from β-NADH. Commercially available preparations contain only 80-90% α-NADH. The anomerization kinetics of α- and β-NADH has been investigated at pH 7.0 and 7.3 taking into account the irreversible conversion reactions of α- and β-NADH. The H + ion activity related anomerization rate constants k 1 (α to β) and k 2 (β to α) have been found to be 170 and 29 M −1 s −1, respectively, at pH 7.0 and 192 and 36 M −1 s −1 respectively, at pH 7.3. The equilibrium constants k 1/ k 2 are 5.8 and 5.3 at pH 7.0 and pH 7.3, respectively. Absorption and fluorescence properties of α-NADH preparations have been determined newly. At 25°C, maximum absorption was found at 260.1 and 346.6 nm, whereas at 37°C the wavelengths were 260.1 and 345.3 nm. Maximum fluorescence excitation was found at 348 nm, and maximum emission was at 453 nm.