  The dynamics of polarized fluorescence in NADH at 460 nm under two-photon
excitation at 720 nm by femtosecond laser pulses in water-methanol solutions
has been studied experimentally and theoretically as a function of methanol
concentration. A number of fluorescence parameters have been determined from
experiment by means of the global fit procedure and then compared with the
results reported by other authors. A comprehensive analysis of experimental
errors was made. The interpretation of the experimental results obtained was
supported by ab initio calculations of the structure of NADH and NMNH in
various solutions. An explanation of the heterogeneity in the measured decay
times in NADH and NMNH has been suggested based on the influence of the
internal molecular electric field in the nicotinamide ring on non-radiative
decay rates. We suggest that different charge distributions in the cis and
trans configurations result in different internal electrostatic field
distributions that lead to the decay time heterogeneity. A slight but
noticeable rise of the decay times tau_1 and \tau_2 with methanol concentration
was observed and treated as a minor effect of a non-radiative relaxation
slowing due to the decrease of solution polarity. The analysis of the
rotational diffusion time tau_r as a function of methanol concentration on the
basis of the Stokes-Einstein-Debye equation allowed for determination of
relative concentrations of the folded and unfolded NADH conformations in
solutions. The analysis of the fluorescence anisotropy parameters and parameter
Omega determined from experiment allowed for determination of the two-photon
excitation tensor components and suggested the existence of two excitation
channels with comparable intensities.
