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

Abstract

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 τ 1 and τ 2 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 xz 2 + S yz 2 | 1/2 .

Cellular NADH and NADPH Conformation as a Real-Time Fluorescence-Based Metabolic Indicator under Pressurized Conditions

“…We compare pressure cycling results with the piezochromic response of NADH solutions. Figure 3 shows spectra from NADH in solutions of varying polarity; methanol (from 0 to 90 vol%) instead of ethanol is used to vary the polarity due to the availability of previous studies on excited-state dynamics in water–methanol mixtures, which indicate an opening of the molecular conformation with increasing methanol [ 35 , 36 , 37 ]. We observe an increase in intensity and a shift to shorter emission wavelength as the methanol concentration is increased.…”

Линейный Дихроизм И Двулучепреломление Зондирующего Излучения В Спектроскопии Накачка-Зондирование В Многоатомных Молекулах

“…Быстрые релаксационные процессы, происходящие в возбужденных состояниях многоатомных молекул в растворах и живых клетках, очень часто являются анизотропными, обычно они исследуются методами многофотонной поляризованной флуоресценции [7][8][9][10][11][12] или методами поляризационной спектроскопии pumpprobe [13][14][15][16][17][18][19][20][21]. В обоих случаях линейно поляризованный лазерный импульс фемтосекундной длительности переводит исследуемые молекулы в возбужденное электронное состояние, причем образующийся ансамбль возбужденных молекул, как правило, характеризуется выстраиванием по направлениям молекулярных осей.…”

Two-Photon Excited Fluorescence Dynamics in Enzyme-Bound NADH: the Heterogeneity of Fluorescence Decay Times and Anisotropic Relaxation

“…Numerous time-resolved fluorescence studies carried out in solutions, cells, and living tissues revealed that after excitation in the first absorption band at 320-370 nm related to the nicotinamide (NA) chromophore group NADH exhibits a multicomponent decay dynamics. In aqueous solution free NADH is known to exhibit biexponential fluorescence decay with the lifetimes of about 0.3 ns and 0.7 ns [14,15,16,17,18,19,20]. In other solvents the lifetimes and corresponding weighting coefficients depend strongly on temperature and solvent type [21,15,14,17,20].…”

“…In aqueous solution free NADH is known to exhibit biexponential fluorescence decay with the lifetimes of about 0.3 ns and 0.7 ns [14,15,16,17,18,19,20]. In other solvents the lifetimes and corresponding weighting coefficients depend strongly on temperature and solvent type [21,15,14,17,20]. At the same time in enzyme-containing solutions and in cells it is most common to fit NADH fluorescence decay in the nanosecond time domain by two exponentials, one of them having the decay times of several ns and another of about 0.4 ns.…”

“…This paper aims to address, at least partly, these important issues. In our recent paper [20] the decay of polarized fluorescence in NADH at 460 nm un-der two-photon excitation at 720 nm was studied in detail in water-methanol solutions as a function of methanol concentration. Based on the analysis of the experimental results obtained and ab initio calculations performed we have suggested that the heterogeneity in the measured decay times in free NADH in solutions can be due to different charge distributions in the cis and trans configurations of the nicotinamide ring that result in different intramolecular electrostatic field distributions and lead to two different non-radiative decay rates.…”