The Effect of Buffers on Nicotinamide Adenine Dinucleotide Hydrolysis

The presence of alkali accelerates the rate of destruction of pyridine nucleotides (I), and the cleavage of the nicotinamideribose linkage of nicotinamide adenine dinucleotide (NAD) under alkaline conditions has also been demonstrated (2). Prolonged treatment in alkali can cause cleavage of the pyrophosphate linkage of NAD as demonstrated by the release of adenylic acid. However, the hydrolysis of this linkage appears to occur after an initial hydrolysis of the nicotinamide-ribose bond has taken place (3). Colowick, Kaplan, and Ciotti (4) observed that the rate of hydrolysis of the nicotinamide-ribose linkage of NAD at 100” increases with increasing pH above pH 6.0. The rates of hydrolysis obtained in various buffers at pH 7.5 were found to depend on the type of buffer used. Tris and acetate buffers did not increase the rate of hydrolysis above that observed with unbuffered NAD at this pH, whereas phosphate and citrate buffers enhanced it 3- to 4-fold. It was noted (1) that Tris could almost completely abolish this stimulatory effect of phosphate and citrate buffers. In a study of the stability of pyridine nucleotides, Lowry, Passonneau, and Rock (5) confirmed the phosphate stimulation of NAD hydrolysis as well as the dependence of this reaction on pH in the region of 6.0 to 8.0. The phosphate-stimulated hydrolysis of NAD has proved to be a convenient procedure for liberating the pyridine moiety from a dinucleotide for purposes of identification (6, 7). One advantage of this chemical hydrolysis over the enzyme-catalyzed cleavage of NAD and related dinucleotides is the freedom from specificity requirements that often arise in the enzyme reactions. The experiments reported here were carried out to elucidate the roles of phosphate and Tris buffers in the hydrolytic reactions with NAD and related compounds.