Two-Photon Excited Fluorescence of NADH-Alcohol Dehydrogenase Complex in a Mixture with Bacterial Enzymes.

Thorough study of composition and fluorescence properties of a commercial reagent of active equine NAD-dependent alcohol dehydrogenase expressed and purified from has been carried out. Several experimental methods: spectral- and time-resolved two-photon excited fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fast protein liquid chromatography, and mass spectrometry were used for analysis. The reagent under study was found to contain also a number of natural fluorophores: free NAD(P)H, NADH-alcohol dehydrogenase, NADPH-isocitrate dehydrogenase, and pyridoxal 5-phosphate-serine hydroxymethyltransferase complexes.

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

The dynamics of polarized fluorescence in NADH in alcohol dehydrogenase (ADH) in buffer solution has been studied using the TCSPC spectroscopy. A global fit procedure was used for determination of the fluorescence parameters from experiment. The interpretation of the results obtained was supported by calculations of the NADH structure.

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.

Anisotropic relaxation in NADH excited states studied by polarization-modulation pump-probe transient spectroscopy.

We present the results of experimental and theoretical studies of fast anisotropic relaxation and rotational diffusion in the first electron excited state of biological coenzyme NADH in water-ethanol solutions. The experiments have been carried out by means of a novel polarization-modulation transient method and fluorescence polarization spectroscopy. For interpretation of the experimental results a model of the anisotropic relaxation in terms of scalar and vector properties of transition dipole moments has been developed based on the Born-Oppenheimer approximation.