Two-Photon Excited Fluorescence Dynamics in NADH in Water-Methanol Solutions: The Role of Conformation States

The dynamics of polarized fluorescence in reduced nicotinamide adenine dinucleotide (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. Ab initio calculations of the structure of NADH in water and methanol and of β-nicotinamide mononucleotide (NMNH) in vacuum have been carried out for clarifying the role of decay time heterogeneity. The main results obtained are as follows. An explanation of the heterogeneity in the measured fluorescence decay times in NADH has been suggested based on the influence of the internal molecular electric field in the nicotinamide ring on nonradiative 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 fluorescence decay times τ₁ and τ₂ with methanol concentration was observed and treated as a minor effect of a nonradiative relaxation slowing due to the decrease in solution polarity. Relative concentrations of the folded and unfolded NADH conformations in solutions have been determined using a new method of analysis of the rotational diffusion time τ_r as a function of methanol concentration on the basis of the Stokes-Einstein-Debye equation. The analysis of the fluorescence anisotropy parameters obtained under linearly and circularly polarized excitation and the parameter Ω has been carried out and resulted in the determination of the two-photon excitation tensor components and suggested the existence of two excitation channels with comparable intensities. These were the longitudinal excitation channel dominated by the diagonal tensor component S_zz and the mixed excitation channel dominated by the off-diagonal tensor components |S_xz² + S_yz²|^1/2.