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Combined studies on fluorescence quantum yield in coenzymes NADH and FAD inwater-methanol, water-ethanol and water-propylene glycol mixtures and ontime-resolved fluorescence of the same molecules under excitation at 450 and355~nm by means of the TCSPC method have been carried out. The dependence ofquantum yield in NADH on alcohol concentration was found to be similar inmethanol and ethanol and different from that in propylene glycol. The behaviorof quantum yield in FAD was found to be almost independent of the type ofalcohol and exhibited a dramatic 5--6 times increase with alcoholconcentration. A model describing molecular excited state relaxation dynamicsin pico- and nanosecond time domain was developed on the basis on the quantummechanical theory and used for analysis of the experimental results. Inparticular, the model provided a new insight into the nature of the DecayAssociated Spectra (DAS) phenomenon. The analysis of the role of pico- andnanosecond quenching in NADH suggested that the picosecond decay in methanoland ethanol likely does not occur through electron transfer in the stackingconfiguration of the nicotinamide and adenine moieties, but through othermechanisms. However in propylene glycol the picosecond quenching in NADHdepended strongly on propylene glycol concentration and sharp increase of themeasured quantum yield was associated with the contribution of both picosecondand nanosecond quenching mechanisms. The picosecond quenching to FAD was foundto depend significantly on the any alcohol concentration suggesting that itgives a profound contribution to the rise of the measured quantum yield withalcohol concentration. This conclusion supports the established mechanism ofthe fluorescence quenching in FAD through electron transfer reaction in the$\pi$-stacked conformation between isoalloxazine and adenine moieties.

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