pH-Dependent Flavin Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Ultraviolet Resonance Raman (UVRR) Spectra at Intracellular Concentration

Abstract

The ultraviolet resonance Raman spectra of the adenine-containing enzymatic redox cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide in aqueous solution of physiological concentration are compared with the aim of distinguishing between them and their building block adenine in potential co-occurrence in biological materials. At an excitation wavelength of 266 nm, the spectra are dominated by the strong resonant contribution from adenine; nevertheless, bands assigned to vibrational modes of the nicotinamide and the flavin unit are found to appear at similar signal strength. Comparison of spectra measured at pH 7 with data obtained pH 10 and pH 3 shows characteristic changes when pH is increased or lowered, mainly due to deprotonation of the flavin and nicotinamide moieties, and protonation of the adenine, respectively.

Keywords: FAD; NADH; UVRR; Ultraviolet resonance Raman; adenine; flavin adenine dinucleotide; nicotinamide adenine dinucleotide; pH dependence; protonation.

Graphical Abstract

Figure 1.

The chemical structures of (a) FAD, (b) NADH, and (c) adenine at neutral pH with numbering of the atoms for assignment.

Figure 2.

UV resonance Raman spectra of 10^–4M FAD, NADH, and adenine as indicated at neutral pH (λ  =  266 nm, 3 × 15 s). Scale bars: FAD and NADH 500 counts, adenine 2000 counts.

Figure 3.

UV resonance Raman spectra of (a) adenine, (b) NADH, and (c) FAD at pH 10, pH 7 and pH 3 as indicated (c = 10^–4M, λ = 266 nm, 3 × 15 s). Scale bars: 500 counts.