Two-Photon Excited Fluorescence Dynamics in NADH in water-methanol solutions: the Role of Conformational States.

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.