Reverse-Phase Liquid Chromatographic Analysis of the Acid-Catalyzed Rearrangement of Nadh, Separation of New Products and an Analysis of the First Two Reactions of the Rearrangement

Abstract

The previously unresolved products of acid-catalyzed rearrangement of NADH have been separated into seven peaks by liquid chromatography on microparticle ODS. The peak with the second highest retention volume was identified as O²,6-B-cyclotetrahydronicotinamide adenine dinucleotide. Based on the order of appearance and disappearance of the peaks, and on their production in the presence of glyceraldehyde-3-phosphate dehy-drogenase (EC 1.2.1.12), four of the peaks have been assigned to specific products in the reaction sequence originally proposed by Oppenheimer and Kaplan [Biochemistry, 13, 4675, 1974]. The three remaining peaks (in addition to AMP) are previously unidentified products of the acid-catalyzed rearrangement of 6-B-cyclotetrahydronicotinamide adenine dinucleotide. The first step in the rearrangement of NADH to 6-B-cyclotetrahydronicotinamide adenine dinucleotide was shown to be dependent on the pH and ionic strength of the buffer, but neither the first product nor any other product incorporated the buffer into its structure.