Stabilityof NADPH: Effectof VariousFactorson the Kineticsof Degradation

Seeking to minimizedegradationof NADPH duringstorage, reagentpreparation,and assays, we investigatedthe effects of pH, temperature,and ionicstrengthas well as the effects of phosphate and acetate. NADH was also included for comparison. Our resultsindicatethat the rate of degradation of NADPH is proportionalmost importantlyto temperature and concentrationsof hydroniumion, but also to concentra-tions of phosphate and acetate. The degradation rate de-creased with increasing ionic strength at neutral pH, but increased slightly at lower pH. NADPH generallyis less stable than NADH under the same conditions.The reaction orders with respect to hydroniumionand anionswere near 1 for NADH degradationreactions,about0.5 for NADPH. Rate constants for NADH and NADPH differedmore at higherpH and lower phosphateand acetate concentrations. AddftlonalK.yphrases:variation, source of N4DH compared Reduced forms of both nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) are coenzymes of many dehydrogenases. Al-though there have been extensive studies concerning stabil-ity of NADH, relatively few have involved NADPH, perhaps because no difference between the two was expected. Howev-er, it is interesting that they are fundamentally different in most biochemical reactions: NADH is oxidized by the respi-ratory chain to generate ATP, whereas NADPH serves as a hydrogen and electron donor in reductive biosynthesis. Both NADH and NADPH are known to be unstable in acidic medium (1-4). However, almost all studies were related to NADH and its model compounds such as 1,3-substituted 1,4-dihydropyridine (1, 2). As a result, little is known about the degradation kinetics, the products, or the spectral changes associated with NADPH degradation, al-though Lowry et al. (3) found NADPH degradation to be about 80 % faster than that of NADH at 30#{176}Cand at pH 2 t