Determination of Fluorescence Quantum Yields and Decay Times of Nadh and Fad in Water-Alcohol Mixtures: The Analysis of Radiative and Nonradiative Relaxation Pathways

Combined studies on fluorescence quantum yield of coenzymes NADH and FAD in water–methanol, water–ethanol and water–propylene glycol mixtures and on time-resolved fluorescence of these molecules under excitation at 450 and 355 nm by means of time-correlated single photon counting (TCSPC) method have been carried out. A novel model that allowed to separate the contributions from several excited state relaxation mechanisms to fluorescence dynamics in the pico- and nanosecond time domains and to fluorescence quantum yield was developed and used for analysis of experimental data. The results indicated the existence of at least two different nonradiative relaxation mechanisms in NADH and FAD: a relatively slow nanosecond and a much faster picosecond ones. The analysis suggested that the increase of NADH fluorescence quantum yield with all alcohols concentration occurred mainly due to relatively slow nanosecond non-radiative and radiative relaxation mechanisms related with the change of conformation distributions, while the picosecond fluorescence quenching was found to have practically the same and conformation-insensitive value in water–monohydric alcohol mixtures. Similar conclusion was made for NADH in water–propylene glycol solutions where however a moderate decrease of the picosecond quenching rate was determined at high propylene glycol concentrations that was attributed to a slowdown of picosecond quenching under high viscosity conditions. In the case of FAD the analysis suggested that the dramatic rise of fluorescence quantum yield with alcohol concentration was mostly due to the conformation-sensitive picosecond quenching. This conclusion supports the known mechanism of the picosecond fluorescence quenching in FAD through electron transfer reaction in the π-stacked conformation between isoalloxazine and adenine moieties. Radiative decay times in NADH and FAD were estimated from experimental data and found to be about 10 ns and 20–30 ns, respectively.