Anisotropic fluorescence decay kinetics in FAD in water-methanol solutions under excitation at 355 and 450 nm

The paper presents the study of anisotropic fluorescence decay of FAD in water-methanol solutions of different concentrations in the range of 0-80%. The fluorescence decay parameters: decay times, corresponding weighting coefficients, fluorescence anisotropy, and rotation diffusion time have been determined from experiment using the TCSPC method as function of methanol concentration and analyzed. The fluorescence kinetics demonstrated doubleexponential decay with two fluorescence decay times of about 2 and 4 ns. The decay times τ₁ and τ₂ were found to be the same under excitation at 450 nm and at 355 nm within experimental error bars and were practically independent of methanol concentration. The fluorescence anisotropy under 450 nm and 355 nm excitation was determined to be about 0.35 and 0.23, respectively indicating that the directions of the excitation transition dipole moments via the first and the second absorption bands differed significantly from each other. Also, the anisotropy was found to be practically independent of methanol concentration. The rotational diffusion time was proportional to the solution viscosity at lower methanol concentrations up to 40%, and reached a plateau at higher concentrations while the solution viscosity dropped down. This behavior was explained due to the increase of FAD solvation in the MeOH-water solution under the condition of FAD unfolding due to the denaturation effect.