The role of microenvironment in the mechanisms of fluorescence decay in free and protein-bound NADH

The influence of solution polarity and viscosity on fluorescence decay parameters in free and protein-bound NADH was studied. The fluorescence in NADH dissolved in water, methanol, ethanol, propylene glycol, and alcohol dehydrogenase-containing solution was two-photon excited by femtosecond laser pulses at 720 nm and recorded by means of the time-correlated single photon counting (TCSPC) method. Fluorescence decay times and corresponding weighting coefficients were determined by fit from polarization-insensitive fluorescence decay experimental signals. The fluorescence decay times τ₁ and τ₂, and weighting coefficients a₁ and a₂ were found to depend significantly on the solution type. A model describing the dependence of the fluorescence decay parameters on the microenvironment in solutions with different polarity and viscosity has been developed. According to the model, the heterogeneity in the measured fluorescence decay times in NADH and the time values were closely related with the charge distributions in the cis and trans configurations of the nicotinamide ring that result in different electrostatic field and different non-radiative decay rates. The influence of solution polarity and viscosity on the measured fluorescence decay times was investigated. As shown in high viscous solutions the increase of fluorescence decay times in NADH was mostly due to slowing down of the nuclei motions during the vibrational relaxation and intramolecular nuclear rearrangement whereas in low viscous solutions the fluorescence decay times follow the change of solution polarity.