Gertz Likhtenshtein

Dual fluorophore–nitroxide probes for analysis of vitamin C in biological liquids

A new method for quantitative analysis of vitamin C in biological and chemical liquids was proposed. The method is based on the use of dual molecule consisting of a fluorescent chromophore and a nitroxide radical. In the dual molecule, the nitroxide acts as a quencher of the fluorescence of the chromophore fragment. Reduction of the nitroxide fragment by ascorbic acid results in decay of ESR signal and enhancement of the fluorescence. By performing the series of pseudo-first-order reactions between the dual molecule and ascorbic acid and consequent plotting rate constants versus ascorbic acid concentrations the calibration curves for the vitamin C analysis were obtained. Variations of chemical structure of fluorophore and nitroxide fragments allow to regulate fluorescent properties and redox potentials of the dual molecules. The proposed fluorophore-nitroxide hybrids retain all features of the spin labels and fluorescence probes gaining new advantages for monitoring redox reactions and radical processes by two independent techniques: ESR and steady-state fluorescent spectroscopy. The method was applied to the vitamin C analysis in commercial fruit juices.

Photophysical characterization of trans-4,4′-disubstituted stilbenes

Abstract Absorption and emission spectra of twenty trans -4,4′-disubstituted stilbenes have been measured in four solvents: cyclohexane (CH), chlorobenzene (CB), 2-butanone (methylethyl ketone, MEK) and dimethylsulfoxide (DMSO) at room temperature. Fluorescence quantum yields ( Φ f ) and fluorescence lifetimes ( τ f ) have been measured for these stilbenes. The lifetimes and quantum yields of fluorescence were found to be dependent on the donor-acceptor properties of the substituents and correlate with the Hammett ν-constants. In addition, we experimentally observed the appearance of a second emitting state which is close energetically to the lowest exited singlet state 1 t ∗ in cases of strong donor-acceptor substituents in polar solvents.

Substituted stilbenes: a new view on well-known systems: New applications in chemistry and biophysics

Abstract In this review, we present new areas of application of stilbene compounds in chemistry and biophysics. The first is the application of the linear free-energy relationships (LFER) to photochemistry of substituted stilbenes. The second is the development of the fluorescence-photochrome technique for investigation of different dynamical processes in biological membranes and surface systems. The various time-scale processes occurring with the stilbene molecules after irradiation were found to exhibit different sensitivity to intramolecular donor–acceptor effects of substituents and medium polarity. The sensitivity is quantitatively characterised by ρ -constant of the linear Hammett-like relationships. The obtained experimental data on LFER has independently confirmed the Saltiel mechanism of the photoisomerisation and established the quantitative basis for detailed mechanistic analysis of the different stages of this important photochemical reaction. The measurements of the direct and sensitised trans – cis and cis – trans photoisomerisation allowed investigation the rotational and translational diffusion of the stilbene labels and their aromatic moieties labelled proteins in a wide temporal range. The cascade photochemical system based on the combination of the stilbene, triplet and nitroxide-spin probes gained an additional advantage to measure the rate constant of the triplet state quenching and to estimate the local concentration of stable radicals. High sensitivity, validity and relatively low cost of conventional fluorescence facilities are the main advantages of these fluorescent-photochrome methods.

Novel Fluorescent Methods for Biotechnological and Biomedical Sensoring: Assessing Antioxidants, Reactive Radicals, NO Dynamics, Immunoassay, and Biomembranes Fluidity

We proposed and developed a series of fluorescent methods for analysis and investigation of biological systems with a view of future biotechnological and biomedical applications. The methods we describe have been built upon several photochemical and photophysical phenomena including fluorescent quenching, photochrome photoisomerization, and energy transfer. Three new types of molecular probes have been developed and employed for such studies: (1) dual fluorophore–nitroxide compounds, (2) fluorescence–photochrome molecules, and (3) super molecules containing both fluorescence and fluorescent quenching segments. The fluorescent properties of the new probes were intensively exploited for several practical applications including a real-time analysis of antioxidants, nitric oxide, superoxide, reactive radicals, trinitrotoluene, and metal ions, investigation of molecular dynamics of biomembranes in a wide range characteristic times, detection of protein conformational transition, and characterization of surface system. Owning high sensitivity, simplicity, and availability of fluorescent techniques, these methods can be widely employed and are adaptable to fibrooptic sensoring. A general survey of the physical principles and application of the new fluorescent methods has been provided.

The reduction of a nitroxide spin label as a probe of human blood antioxidant properties.

The kinetics of reduction of the radical R ”, 5-dimethylaminonaphthalene-1-sulfonyl-4-amino-2,2,6,6-tetramethyl-1-piperidine-oxyl, by blood and its components were studied using the EPR technique. The results demonstrate that R ” is adsorbed to the outer surface of the membrane and does not penetrate into the erythrocytes. A series of control experiments in PBS demonstrate that ascorbate is the only natural reducing agent that reacts with R ” . The observed first order rate of disappearance of the nitroxide radical, k , is: k blood > k eryth > k plasma and k blood ; k eryth + k plasma . The results demonstrate that: The erythrocytes catalyze the reduction of R ” by ascorbate. The rate of reduction of the radical is high though it does not penetrate the cells. In human erythrocytes there is an efficient electron transfer route through the cell membrane. The study points out that R ” is a suitable spin label for measuring the reduction kinetics and antioxidant capacity in blood as expressed by reduction by ascorbate.

Novel fluorescence-photochrome labeling method in the study of biomembrane dynamics

A novel photochrome-fluorescence method (PFLM) based on monitoring fluorescence parameters and kinetics of photochrome photoisomerization of para-substituted stilbenes (PSS) has been proposed. It was shown that PSS exhibits fluorescence characteristics which are similar to ones of typical membrane fluorescence probes such as diphenylhexatriene (DPH). A study of kinetics of PSS trans-cis and cis-trans photoisomerization makes it possible to estimate, under certain conditions, the rotational correlation time of the stilbene fragments in the excited state of PSS for the fixed angle 180 degrees. In viscous media this process is a rate-determining stage. Taken together, the both techniques, fluorescence and photochrome, make it possible to establish a detailed mechanism and measure quantitative parameters of stilbene probe (PSS) mobility in a membrane. The PFLM was applied to the study of E. coli membrane dynamics.

Effect of albumin on the kinetics of ascorbate oxidation

The fluorescence intensity of the fluorophore in dansyl piperidine-nitroxide is intramolecularly quenched by the nitroxyl fragment. Therefore, the oxidation of ascorbic acid by the fluorophore-nitroxide (FN) probe can be monitored by two independent methods: steady-state fluorescence and electron paramagnetic resonance. Bovine serum albumin (BSA) affects the rate of this reaction. The influence of BSA on the rate is attributed to the adsorption of both ascorbate and the probe to BSA. Adsorption of ascorbate to BSA is confirmed by NMR relaxation experiments. The spatial distribution of the molecules on the BSA surface changes the availability of ascorbate and FN to each other. The results also point out that, in the presence of BSA, the autoxidation of ascorbate is significantly slowed down. The effect is studied at different pH values and explained in terms of the electrostatic interaction between the ascorbate anion and the BSA molecule.

Factors affecting photoinduced electron transfer in a donor—acceptor pair (DA) incorporated into bovine serum albumin

Abstract A donor—acceptor hybrid molecule (DA) consisting of a 1-dimethyl-aminonaphthalene-5-sulphonate group in an excited singlet state (donor) and a nitroxide radical (acceptor) was incorporated into a hydrophobic cavity of bovine serum albumin (BSA). The kinetics of reversible and irreversible intramolecular electron transfer from the donor to the acceptor were monitored by luminescence and ESR techniques in the temperature range from 77 K to 300 K. The temperature dependence of the micropolarity and the intramolecular dynamics in the vicinity of the donor and the acceptor groups were monitored by fluorescence and ESR techniques, respectively. The Arrhenius dependence of the reversible ET constant ( K e was non-linear and the apparent activation energy ( E app changes from 0 eV (at T =77–100 K) to 0.25 eV (at T =298 K). The temperature region of the E app increase (100–240 K) was close to the temperature of the increase of relaxation shift of the donor luminescent spectra ( T = 100–240 K). On the basis of the obtained kinetic data and of the data of the micropolarity in the vicinity of the donor and the acceptor groups, values of standard Gibbs energy (ΔG 0 ) change, the reorganization energy ( E r , the resonance integral ( V DA ) and the Frank—Condon factor for reversible electron transfer were estimated. The role of molecular dynamics in the vicinity of the donor and the acceptor groups is also discussed.

Role of orbital overlap and local dynamics in long-distance electron transfer in photosynthetic reaction centres and model systems

Abstract The following approaches have been developed to estimate the factors affecting electron transfer (ET) in proteins: (1) an analysis of the correlation between the rate constants of ET ( k ET ) in a donor-acceptor (DA) pair and the dynamic parameters of various modes of relaxation of media traced by spin, fluorescent, triplet and Mossbauer labelling methods; (2) the use of spin exchange (SE) data in biradicals and complexes of transition metals with paramagnetic ligands and triplet-triplet energy transfer (TTET) data, which are considered as simple models of ET, for the estimation of the attenuation parameter ( γ X ) of superexchange through an individual chemical group X and through homogeneous “conducting” and “non-conducting” media. The application of the aforementioned approaches to ET in photosynthetic reaction centres (RCs) and DA pairs in simple liquids and proteins is reviewed. The mechanism of ET in the systems under consideration is discussed. A new procedure of analysis of kinetic-thermodynamic relationships, including predictions by Marcus theory, is proposed.

Quenching of the cascade reaction between triplet and photochrome probes by nitroxide radicals

We proposed a new method for the study of molecular dynamics and fluidity of the living and model biomembranes and surface systems. The method is based on the measurements of the sensitized photoisomerization kinetics of a photochrome probe. The cascade triplet cis-trans photoisomerization of the excited stilbene derivative sensitized with the excited triplet Erythrosin B has been studied in a model liposome membrane. The photoisomerization reaction is depressed with nitroxide radicals quenching the excited triplet state of the sensitizer. The enhanced fluorescence polarization of the stilbene probe incorporated into liposome membranes indicates that the stilbene molecules are squeezed in a relatively viscous media of the phospholipids. Calibration of the “triple” cascade system is based on a previously proposed method that allows the measurement of the product of the quenching rate constant and the sensitizer’s triplet lifetime, as well as the quantitative detection of the nitroxide radicals in the vicinity of the membrane surface. The experiment was conducted using the constant-illumination fluorescence technique. Sensitivity of the method using a standard commercial spectrofluorimeter is about 10−12 mol of fluorescence molecules per sample and can be improved using an advanced fluorescence technique. The minimal local concentration of nitroxide radicals or any other quenchers being detected is about 10−5 M. This method enables the investigation of any chemical and biological surface processes of microscopic scale when the minimal volume is about 10−3 µL or less.