Anatoly Nikolaevich Rubinov

Site selectivity in excited-state intramolecular proton transfer in flavonols

Abstract To investigate 4′-(diethylamino) (FET) and 4′-N-(15-azacrown-5) (FCR) derivatives of 3-hydroxyflavone in binary solvents and erythrocyte ghosts, we used the red-edge excitation spectroscopy (REES). The results obtained prove the existence of spectral heterogeneity of flavonols in the studied systems. The effect manifests itself in the dependence of the efficiency of excited-state intramolecular proton transfer (ESIPT) on the excitation frequency. The electro-optical absorption method (EOAM) was used to measure the dipole moments of the normal form of FET. The electric dipole moments in the ground (μg) and excited Franck–Condon ( μ e FC ) states have the values 22.7×10−30 and 53.3×10 −30 C m , respectively. On optical excitation, the electric dipole moment increases by 34×10 −30 C m , and the angle between μg and μ e FC is 25°. The results of the electro-optical and spectroscopic measurements enable us to describe more precisely the process of charge and proton transfer in 4′-amino-3-hydroxyflavones. Charge transfer and proton transfer occur alternately. The main stage of forward electron charge transfer takes place after excitation of the normal form (N∗) and partly after ESIPT. In the phototautomer (T), only partial reverse charge transfer happens after photon emission. The second, more efficient stage of reverse electron charge transfer occurs after radiationless conversion of phototautomer into the normal form.

Selective laser spectroscopy of 1-phenylnaphthylamine in phospholipid membranes.

Time-resolved and steady-state spectra and kinetics of anisotropy of 1-phenylnaphthylamine (1-AN) fluorescent probe in phosphatidylcholine bilayer membranes have been examined using a nanosecond laser spectrofluorimeter. In this system we consider two kinds of inhomogeneous broadening of spectra, the first of which is due to different probe locations in membrane, while the second one is due to the statistical distribution of interaction energy within a given location. This broadening causes the red shift of the fluorescence spectrum at red-edge excitation, the specific dependences of instantaneous fluorescence and fluorescence anisotropy spectra on the wavelength of excitation. A field diagram is presented which, by describing the free energy levels of the polar fluorescent probe in membranes, makes it possible to unambiguously interpret the whole set of experimental data. It is suggested that the release of potential energy of intermolecular interactions which occurs in the process of relaxation, results in accelerated (light-induced) rotation of the probe inside the membrane.

Dipole moments of 4'-aminoflavonol fluorescent probes in different solvents.

Electrooptical absorption measurements (EOAM) were used to measure the dipole moments of the normal form of 4′-(dimethylamino)-3-hydroxyflavone (FME), and 4′ N-(15-azacrown-5)-3-hydroxyflavone (FCR) in 1,4-dioxane, toluene, and cyclohexane. With these probes excited-state intramolecular proton transfer (ESIPT) takes place. For comparison, the dipole moments of 4′-(dimethylamino)-3-metoxyflavone (FME3ME), for which ESIPT is lacking, were measured, too. For all three probes the ground (μg) and excited Franck-Condon state (μeFC) electrical dipole moments are parallel to each other and also parallel to the transition dipole moment. The electrical dipole moments of FCR, FME, and FME3ME in their ground state have values within the range (12.0–17.7) × 10−30 C m. Upon optical excitation, the dipole moments increase by (41.9–52.9) ×10−30 C m. The value of the change of the dipole moment vector Δaμ with excitation to the Franck-Condon state and the value of the vector μeFC for FCR and FME are practically independent on the solvent polarity. From this point of view and due to large values of the dipole moments FCR and FME are very promising probes for the investigation of the distribution of the local polarity in biological systems using site-selective excitation of the different sites. Our steady-state fluorescence studies on FME and FCR have demonstrated a high spectral sensitivity of the normal form to such solvent characteristics as polarity.

Fluorescence decay time distribution for polar dye solutions with time-dependent fluorescent shift

Abstract A new method for the recovery of the fluorescence decay time distributions based on the combination of a truncated singular value decomposition and computation of the solution in the presence of non-negative constraints is described. The shape of the fluorescence decay time distribution of polar dye solutions with inhomogeneous broadening of the electronic levels, due to fluctuations of dipole-dipole interactions, was analyzed both theoretically and experimentally. For the first time it has been found that under conditions of single- or double-exponential time-dependent fluorescent shift the distribution is not continuous but consists of discrete peaks. The positions and weights of these peaks are determined by the parameters of the spectral shift. A method for the determination of the dynamic characteristics of the polar region in the membrane bilayer is proposed based on decay time distribution analysis.

Wavelength-dependent rotation of dye molecules in a polar solution.

Investigation of rotation movement of 3-amino-N-methylphthalimide in glycerol was carried out, taking into consideration the fluctuation of solvate structure. It was shown theoretically and experimentally that structural relaxation of the solvate shell, which follows excitation of the dye molecule, causes not only shift of the fluorescence spectrum in time but also additional rotation of the dye molecule. This effect, which may be called “wavelength-dependent rotation”, depends on the light frequency of both excitation and fluorescence. In particular, at excitation near the maximum of the absorption band, when the relaxation process is followed with the red shift of the fluorescence maximum, the anisotropy of fluorescence decreases faster in the red part of the fluorescence band than in the blue part. On the contrary, in the case of far anti-Stokes excitation, when the temporal shift of fluorescence is going to the blue, the anisotropy in the red part of the spectrum drops more slowly than in the blue part. Finally, there is a special excitation frequency which causes neither change of the fluorescence maximum nor acceleration of the rotational movement of the dye molecule. It is also shown that the temporal evolution of the spectrum and anisotropy of fluorescence in a polar dye solution may be quantitatively described using the socalled inhomogeneous broadening function (IBF). This function gives the distribution of dye molecules in a solution over frequencies of pure electronic transition due to fluctuations of the surrounding shell structure. Measurements of IBF changes in time carried out for 3-amino-N-methylphthalimide showed that during first 3 ns after excitation, the half-width of the IBF grows, and at the same time its maximum quickly shifts to the red. At the later time period there are only small changes of IBF position but considerable exponential decrease in its half-width. The IBF during this period preserves the Gaussian shape.

Effect of the External Refractive Index on Fluorescence Kinetics of Perylene in Human Erythrocyte Ghosts

Fluorescence kinetics of perylene molecules in hemoglobin-free human erythrocyte membranes is investigated as a function of the refractive index of the external medium varied by adjusting the concentration of sorbitol or sucrose in an aqueous suspension of erythrocyte ghosts. It has been found that the fluorescence of perylene in erythrocyte ghosts decays nonexponentially, with the mean decay time decreasing from 7.13 to 5.70 ns with an increase in the refractive index of the suspension from 1.333 to 1.442. An analysis of the dependence made it possible to obtain an estimate of the second-rank orientational order parameter of perylene in the human erythrocyte membrane «P2 (cosθ)» = 0.32 ... 0.43, which bears witness of considerable ordering of perylene molecules along acyl chains of phospholipids constituting the membrane. Good correspondence of the order parameter with the value of the steady-state emission anisoptropy of perylene in erythrocyte ghosts suggests that acyl chains of phospholipids in the human erythrocyte membrane are predominantly oriented along the normal to its surface.

Automated laser spectrofluorimeter for biology amd medicine

Here we wish to present automated laser spectrofluorimeter and some applications of it. The excitation part of the spectrofluorimeter includes an atmospheric pressure nitrogen laser (pulse duration 0.35 ns, peak power 350 kW) and distributed-feedback dye laser (tuning range 400 - 750 nm, spectral width 0.1 - 0.6 nm). It is possible to excite the probe by nitrogen laser or dye laser. The signal of fluorescence is registered by boxcarintegrator and data are input into the computer IBM PC/AT-286. Laser spectrofluorimeter is full automated, lasers, all optical and electronic systems are controlled by the computer. The laser spectrofluorimeter can measure time-resolved fluorescence characteristics with temporal resolution from subnanoseconds to milliseconds: (1) instantaneous fluorescence spectra (0.2 ns - 10 ms), (2) instantaneous anisotropy spectra and anisotropy kinetics (0.2 ns - 10 ms), (3) fluorescence kinetics and lifetime of excited state (0.05 ns - 10 ms). Experimental data can be treated with a full set of programs: (1) deconvolution of fluorescence pulse with up to 3 exponential approximation, (2) calculation of anisotropy kinetics with simultaneous deconvolution for different polarization, (3) calculation of instantaneous spectra from fluorescence decays, (4) mathematical treating of spectra (position of spectrum, halfwidth, asymmetry etc.).

Ultrafast time-resolved fluorescence spectroscopy of novel ketocyanine dyes

Using ultrafast time-resolved fluorescence spectroscopy, the fluorescent characteristics of two ketocyanine dyes—2,5-di[(E)-1-(4-diethylaminophenyl)methylidene]-1-cyclopentanone (CPET) and 2,5-di⨑ub;(E)-1-[4-(4,7,10,13-tetraoxa-1-azacyclopentadecyl)phenyl] ???methylidene⫂ub;-1-cyclopentanone (CPCR)—are studied in organic solvents and in membranes of human erythrocyte ghosts. The position of the fluorescence spectrum of each probe depends strongly on the polarity of the environment due to a considerable change in the dipole moment of the probe after its excitation. An inhomogeneous broadening of the electronic spectra of both dyes is observed, which manifests itself in the bathochromic shift of the fluorescence spectrum upon displacement of the excitation frequency toward the red edge of the absorption spectrum, as well as in the dependence of the shape of the excitation spectrum on the frequency of recording. The lifetime of the excited state of the probes increases with increasing polarity or viscosity of the environment. Analysis of the instantaneous emission spectrum of CPET and of its fluorescence decay time distribution in human erythrocyte ghosts shows that there are two types of binding sites of the probe to the lipid membrane.

Time-resolved fluorometry of red blood cell membrane conformational changes in a population exposed to low-dose radiation

Using subnanosecond laser spectrofluorometry the spectral and polarization time-resolved characteristics of 1-phenylnaphthylamine (1-AN) fluorescent probe in phospholipid bilayer and red blood cell (RBC) membranes have been studied. It is shown that the electronic spectra of probe in model membranes inhomogeneously broadened. Inhomogeneous broadening affect significantly the spectroscopic properties of probe in membranes, such as the time-dependent fluorescence Stokes shift, the dependence of the fluorescence instantaneous spectra and fluorescence kinetics on excitation wavelength. The obtained results prove the existence of the earlier unknown effect of the wavelength- dependent rotation of a probe in phospholipid bilayer and RBC membranes.

Red-edge excitation effect in intramolecular proton transfer in flavonols

To investigate of 4-diethylamino (FET) and 4-(15-azacrown- 5) (FCR) derivatives of 3-hydroxyflavone in binary solvents and erythrocyte ghosts, we used the red-edge excitation spectroscopy. The results obtained prove the existence of spectral heterogeneity of flavonols in the studied systems. The effect manifests itself in the dependence of the efficiency of excited-state intramolecular proton transfer on the excitation frequency.