Determination, purification, and characterization of alpha-NADH.
alpha-NADH can be determined in the presence of high concentrations of beta-NADH using anion exchange HPLC combined with oxidation of beta-NADH by lactate dehydrogenase. The method is suited for the detection of a large number of impurities. Residual absorption is a poor measure of alpha-NADH. By combination of preparative anion exchange chromatography and reversed-phase HPLC, alpha-NADH can be prepared with 95-98% purity starting from beta-NADH. Commercially available preparations contain only 80-90% alpha-NADH. The anomerization kinetics of alpha- and beta-NADH has been investigated at pH 7.0 and 7.3 taking into account the irreversible conversion reactions of alpha- and beta-NADH. The H+ ion activity related anomerization rate constants kappa 1 (alpha to beta) and kappa 2 (beta to alpha) 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 kappa 1 /kappa 2 are 5.8 and 5.3 at pH 7.0 and pH 7.3, respectively. Absorption and fluorescence properties of alpha-NADH preparations have been determined newly. At 25 degrees C, maximum absorption was found at 260.1 and 346.6 nm, whereas at 37 degrees 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.
Klemm
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A
Institute of Biochemistry, Faculty of Medicine, Friedrich Schiller University, Jena, Germany.
Steiner
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T
Cumme
G A
GA
Horn
A
A
D000838:Anions; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005470:Fluorometry; D007700:Kinetics; D009243:NAD / Q000737:chemistry / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013237:Stereoisomerism
D000838:Anions; D009243:NAD