The purification of nicotinamide adenine dinucleotide and kinetic effects of nucleotide impurities.

Large variations of the maximal rate of aldehyde reduction by liver alcohol dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH) showed that commercial preparations of this coenzyme contain variable amounts of an inhibitor (1). The observed kinetic effects were reproduced quantitatively by a known competitive inhibitor, adenosine diphosphate ribose, added in constant molar ratio to the coenzyme, and were consistent with theoretical treatment of this unfamiliar situation (2, 3). It was evident from the theory that in the reverse reaction, small amounts of inactive nucleotide impurity in the oxidized coenzyme, NAD+, might cause large errors in the estimation of the Michaelis constant for the substrate alcohol, in neutral and acid solution. Such errors would explain the deviations of initial rate data for liver alcohol dehydrogenase from the requirements of a compulsory order mechanism, first reported some years ago (4, 5) and recently confirmed by more detailed and precise measurements (6). The detection and separation of such an inhibitor from several commercial preparations of NAD+ of high purity and the general kinetic effects of competing impurities in coenzymes, especially in relation to tests of mechanism, are described in this paper. Kinetic evidence of the mechanism of liver alcohol dehydrogenase will be reconsidered in the light of these findings in a later paper.r Preliminary accounts of aspects of this work have been published (7, 8).