Raman Spectroscopy of Oxidized and Reduced Nicotinamide Adenine Dinucleotides

We have measured the Raman spectra of oxidized nicotinamide adenine dinucleotide, NAD⁺, and its reduced form, NADH, as well as a series of fragments and analogues of NAD⁺ and NADH. In addition, we have studied the effects of pH as well as deuteration of the exchangable protons on the Raman spectra of these molecules. In comparing the positions and intensities of the peaks in the fragment and analogue spectra with those of NADH and NAD⁺, we find that it is useful to consider these large molecules as consisting of component parts, namely, adenine, two ribose groups, two phosphate groups, and nicotinamide, for the purpose of assigning their spectral features. The Raman bands of NADH and NAD⁺ are found generally to arise from molecular motions in one or another of these molecular moieties, although some peaks are not quite so easily identified in this way. This type of assignment is the first step in a detailed understanding of the Raman spectra of NAD⁺ and NADH. This is needed to understand the binding properties of NADH and NAD⁺ acting as coenzymes with the NAD-linked dehydrogenases as deduced recently by using Raman spectroscopy.