Determination of quantum yield of NADH and FAD in alcohol-water solutions: the analysis of radiative and nonradiative relaxation pathways

Combined studies on fluorescence quantum yield in coenzymes NADH and FAD in water-methanol, water-ethanol and water-propylene glycol mixtures and on time-resolved fluorescence of the same molecules under excitation at 450 and 355~nm by means of the TCSPC method have been carried out. The dependence of quantum yield in NADH on alcohol concentration was found to be similar in methanol and ethanol and different from that in propylene glycol. The behavior of quantum yield in FAD was found to be almost independent of the type of alcohol and exhibited a dramatic 5--6 times increase with alcohol concentration. A model describing molecular excited state relaxation dynamics in pico- and nanosecond time domain was developed on the basis on the quantum mechanical theory and used for analysis of the experimental results. In particular, the model provided a new insight into the nature of the Decay Associated Spectra (DAS) phenomenon. The analysis of the role of pico- and nanosecond quenching in NADH suggested that the picosecond decay in methanol and ethanol likely does not occur through electron transfer in the stacking configuration of the nicotinamide and adenine moieties, but through other mechanisms. However in propylene glycol the picosecond quenching in NADH depended strongly on propylene glycol concentration and sharp increase of the measured quantum yield was associated with the contribution of both picosecond and nanosecond quenching mechanisms. The picosecond quenching to FAD was found to depend significantly on the any alcohol concentration suggesting that it gives a profound contribution to the rise of the measured quantum yield with alcohol concentration. This conclusion supports the established mechanism of the fluorescence quenching in FAD through electron transfer reaction in the $\pi$-stacked conformation between isoalloxazine and adenine moieties.