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The spontaneous reactions of reduced nicotinamide-adenine dinucleotide (NADH, DPNH) with orthophosphates in neutral watery milieu were analyzed kinetically. They can be described by three consecutive second-order (pseudo-first-order) reactions. A graphical procedure, suitable for this kinetic analysis, was based on the observation that kl >> k2 >> k?. With the aid of this kinetic analysis it was shown that the reaction most probably involves three consecutive direct transfers of protons from the polybasic anions, which in this case act as proton donors, to the 1,3-substituted 1 ,4-dihydropyridine. Proton transfer W e previously reported changes in the ultraviolet absorption spectra of nicotinamide-adenine dinucleotide (NAD)' in the presence of orthophosphates and certain other anions (Ungar and Alivisatos, 1961). In an extension of our observations, we recently studied the spontaneous reactions of NADH with dihydrogen phosphate and other "polybasic anions" (Stock et al., 1961;Alivisatos et a!., 1964b). The products of these primary interactions react further with the acid anions, in a series of consecutive second-order reactions. A detailed kinetic analysis of these processes, involving graphical evaluation of spectrophotometric data, became necessary. A description of this graphical analysis

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