Evidence against the Natural Occurrence of α-Nicotinamide Adenine Dinucleotide in Azotobacter vinelandii

Extraction of NAD+ and NADH from Azotobacter vinelandii under conditions which do not promote anomerization, indicates that only the β-anomers of NAD+ and NADH are present. No α-NAD+ or α-NADH (measured as α-NAD+ following chemical oxidation) could be found using a chromatographic technique that can separate the β- and α-anomers of NAD+ and detect α-NAD+ at a level of 1% that of β-NAD+. Although there is no detectable α-NAD+ or α-NADH in A. vinelandii, β-NADH does exist in chemical equilibrium with α-NADH (Keq = [α-NADH]/[β-NADH] ≈ 0.18), and this equilibrium is readily established at elevated temperatures (100°) under neutral or mildly alkaline conditions. The data presented indicate that previous reports of α-NAD+ in A. vinelandii and other biological sources are the result of extraction conditions which promote the anomerization of β-NADH to α-NADH. Formation of α-NAD+ occurs by oxidation of α-NADH by various means, among which is the spontaneous transfer of hydrogen from α-NADH to β-NAD+.