Microenvironmental effect on NADH polarized fluorescence studied by ultrafast spectroscopy

Abstract

Nicotinamide adenine dinucleotide (NADH) is an important biological cofactor essential to regulate redox reactions of metabolic pathways in living cells. Nowadays fluorescence kinetic in NADH is actively used for label-free investigation changes in the cell metabolic pathways. As known [1,2] fluorescence decay in NADH has complicated behavior and typically exhibits two decay times. Despite of a large number of studies the nature of this heterogeneity is still not well understood. In this paper we studied the decay of polarized fluorescence in NADH in water/methanol solutions. The dependences of the fluorescence lifetimes, rotational diffusion time, pre-exponential factors, and fluorescence anisotropy on methanol concentration were analyzed and compared with the results reported earlier elsewhere.

The experimental procedure used was similar to that described in detail in our previous papers [2]. Briefly, NADH in water/methanol solutions at the concentration of 1 mM was two-photon excited with femtosecond pulses at 720 nm. Linear and circular polarizations of the laser beam were used in experiments and NADH fluorescence was collected in the direction perpendicular to the laser beam propagation. Two orthogonally polarized fluorescence components were separated by a Glan prism and simultaneously and independently detected by two fast avalanche photodiodes. The signals recorded were analyzed by a time-correlation single photon counting (TCSPC) system. The solutions with different viscosity and polarity used in experiments were prepared by mixing water with methanol at various proportions. The processing of experimental signals was carried out upon convolution of IRF(t) and the fluorescence decay curves by global fit. As a result, fluorescence decay times τ1 and τ2, the ratio of exponential coefficients a2/a1, rotational diffusion time τrot, and anisotropies rl and rc were determined.

The fluorescence decay times τ1 and τ2 was shown increase slowly with methanol concentrations. The ratio a2/a1 was found to be the same at different methanol concentration within the experimental errors, and has the value of 0.28 ±0.1. Non-linear relationship of the rotational diffusion time on methanol concentration was observed with a maximum at about 60%. The anisotropies rl and rc also did not depend on methanol concentration. The fluorescence decay times values are consistence with previously obtained in water [1,2], however there found to be a disagreement with the results obtained previously in methanol [3] that is analyzed in the paper. The observed behavior of the rotational diffusion time τrot was predominately associated with a solution viscosity change and analyzed of the basis of Einstein-Stokes equation [4] where the change of the NADH Stokes volume was also taken into consideration. We suggest that the change in NADH Stokes volume can be related to the change of conformational composition of the coenzyme. The ratio of weight coefficients a2/a1 obtained was analyzed and compared with the results earlier reported elsewhere [1,4]. As shown in the paper, the a2/a1 values determined from the fit are highly sensitive to the shape of an instrumental response function (IRF) that therefore should be recorded with the best possible accuracy.
Considering the nature of two lifetimes in NADH solutions one has to take into account that NADH always exists as an equilibrium ensemble of conformations that can be changed in solutions of different viscosity and polarity. In the paper, we suggested a physical model qualitatively describing the behavior of the lifetimes τ1 and τ2 and the ratio of coefficients a2/a1.

[1] Blacker T. S., Nicolaou N., Duchen M.R., Bain A.J. “Polarized Two-Photon Absorption and Heterogeneous Fluorescence Dynamics in NAD(P)H”, J. Phys. Chem. B, Vol 123, No. 22, pp. 4705-4717, 2019
[2] Vasyutinskii O.S., Smolin A.G., Oswald C., Gericke K.H., “Polarized Fluorescence in NADH under Two-Photon Excitation with Femtosecond Laser Pulses” Opt. and Spec., vol. 122 No. 4, pp. 622-626, 2017.
[3] Ladokhin A. S., Brand L., “Evidence for an excited-state reaction contributing to NADH fluorescence”, Journal of Fluorescence, vol. 5 No. 1, pp. 99-106, 1995.
[4] Blacker T.S., Marsh R.J., Duchen M.R., Bain A.J., "Activated barrier crossing dynamics in the non-radiative decay of NADH and NADPH", Chem. Phys, No. 422, pp. 184-194, 2013.

Speaker

Ioanna A. Gorbunova
Ioffe Institute
Russia

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