[Phosphorescence quenching as an approach for estimating localization of triplet label in cotton fibers].

The method based on the qualitative investigation of chromophore fluorescence (phosphorescence) quenching for instance, by stable nitroxide radical was first used to measure the depth of immersion of triplet label in cotton fiber as a molecular object. The concept of dynamic quenching of fluorescence in solutions and the empirical dependence of the parameters of static quenching between centers with fixed distances were used. The erythrosine triplet labels were incorporated in cotton fibers with subsequent measurement of the efficiency of label phosphorescence quenching and determination of temperature dependence of phosphorescence duration. Using above mentioned approach it became possible for the first time to estimate the depth of immersion of chromophore fragment of the labels (7.4-7.8 A) and study their molecular dynamics in the millisecond range of correlation times. Subtle differences in microstructure and molecular dynamics of the investigated samples were revealed. The proposed approach can be used for investigation of widespread biological and nonbiological objects.

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) approximately = k(eryth) + k(plasma). The results demonstrate that: a. The erythrocytes catalyze the reduction of R* by ascorbate. b. The rate of reduction of the radical is high though it does not penetrate the cells. c. In human erythrocytes there is an efficient electron transfer route through the cell membrane. d. 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.

Stilbene photochrome-fluorescence-spin molecules: covalent immobilization on silica plate and applications as redox and viscosity probes.

We report herein on the development of a new photochrome-fluorescence-spin method for the quantitative analysis of the redox status and viscosity of a medium. The method of the viscosity measurement is based on the use of double fluorescence-nitroxide molecules. In such hybrid compounds the nitroxide moiety quenches the fluorescence of the fluorophore (stilbene moiety). The reduction of nitroxide by an antioxidant (ascorbic acid) causes a rise of fluorescence of the fluorophore. The rate constant of the stilbene fragment photoisomerization in such systems is dependent upon the viscosity of the media. The synthesized dual stilbene-nitroxide probe was covalently immobilized onto the surface of a quartz plate as an eventual fiber-optic sensor. The immobilization procedure included a cyanogen bromide surface activation followed by smoothing with a protein tether. The rate of fluorescence change was monitored in aqueous-glycerol solutions of different viscosities and content of ascorbic acid. Good correlation was found: (a) between the concentration of ascorbic acid in the sample and the rate of fluorescence increase due to the reduction of the nitroxide moiety, and (b) between the rate constant of photoisomerization and the viscosity of the media. Appropriate calibration would make the determination of the viscosity of a media possible (in a range 1-500 cP), as well as ascorbate content, in a range (1-9) x 10(-4) M, with fast single measurement.

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.

Depth of immersion of fluorescent chromophores in biomembranes studied by quenching with nitroxide radical.

A novel method has been developed for measuring depth of immersion of a fluorescent chromophore in biological matrices, such as biomembranes. The method is based on dynamic quenching of chromophore fluorescence by a nitroxide probe freely diffused in solution. Theoretical considerations and experimental evidences relating to the method are discussed. The proposed method was applied to investigation of lecithin liposomes and membranes from Bacillus subtilis modified by the photochrome-fluorescent probe 4,4'-dimethylaminocyanostilbene.

Intramolecular dynamics and conformational transition in proteins studied by biophysical labelling methods. Common and specific features of proteins from thermophylic micro-organisms.

A general survey is carried out on the theoretical grounds for methods of spin, luminescence and Mössbauer labels, as well as their application in the study of protein intramolecular dynamics. When combined, these methods allow the protein dynamics to be investigated within a wide range of correlation times (tau c = 10(2) - 10(-10) s) and amplitudes. The purposeful application of the methods to various proteins at different temperatures (30-330 K), water content, substrate addition, etc., revealed a number of dynamical processes and conformational transitions in proteins. The experiments indicated correlations between the local segmental mobility of protein globules in a nanosecond temporal scale and biochemical reactions, such as long-distance electron transfer, hydrolysis and photoreactions. The biophysical labelling methods results were analysed together with the data on dynamics obtained using complementary physico-chemical methods and theoretical calculations. Special emphasis is given to recent results on proteins from thermophylic micro-organisms. The mechanisms of protein intramolecular dynamics and their role in the stability and functions of proteins and enzymes are discussed.

Quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals. A novel labeling method in study of membranes and surface systems.

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) microL or less.

NMR studies of electrostatic potential distribution around biologically important molecules.

A new experimental approach has been developed to study the distribution of local electrostatic potential around specific protons in biologically important molecules. The approach is the development of a method denoted as "spin label/spin probe," which was proposed by one of us (. Mol. Biol. 6:498-507). The proposed method is based upon the quantitative measurement of the contribution of differently charged nitroxide probes to the spin lattice relaxation rate (1/T1) of protons in the molecule of interest, followed by calculation of local electrostatic potential using the classical Debye equation. In parallel, the theoretical calculation of potential distribution with the use of the MacSpartan Plus 1.0 program has been performed. Application of the method to solutions of simple organic molecules (aliphatic and aromatic alcohols, aliphatic carboxylates (propionate anion), and protonated ethyl amine and imidazole) allowed us to estimate the effective potential around the molecules under investigation. These were found to be in good agreement with theoretically expected values. This technique was then applied to zwitterionic amino acids bearing neutral and charged side chains (glycine, lysine, histidine, and aspartic acid). The reliability of the general approach is proved by the data presented in this paper. Application of this new methodology can afford insight into the biochemical significance of electrostatic effects in biological systems.

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.

[Model molybdenum-sulfur and molybdenum-iron-sulfur protein complexes]. / Model'nye molibdensernye i molibdenzhelezosernye belkovye kompleksy.

A procedure was developed for synthesizing complexes of polynuclear molybdenum sulfide and iron-molybdenum sulfide on the basis of human serum albumin. EPR showed that molybdenum and iron atoms formed clusters in the synthesized complexes. The catalytic activity of the complexes, as determined through reaction of acetylene reduction by sodium borohydride, was significantly higher than that for previously described nonbiological systems, but lower than the characteristic values of FeMo-cofactor of nitrogenase.

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.

[Statistical analysis of electron microphotographs of bioobjects, labelled by electron-dense mercarbide markers]. / Statisticheskii analiz élektronnykh mikrofotografii bioob''ektov, mechennykh élektronno-plotnymi merkarbidnymi metkami.

A method of statistical processing of electron micrographs of molecular objects modified by electron-dense labels containing mercury is proposed. The method allows one to study size, degree of modification and heterogeneity of objects. Application of the method for study of modified nitrogenase and its Fe-Mo containing co-factor, lysozyme, myoglobin, sodium thiomolybdate and trichlortriazine has shown the features of chemical modification and electron micrographs of these molecules. The method can be used for processing of data about complex biological objects modified by electron-dense labels.

[Interaction of spin labels and probes with Na+,K+-ATPase]. / Issledovanie vzaimodeistviia spinovykh metok i zondov s Na+,K+-ATPazoi.

Localization of the PCMB-R spin label and benzocarboline probe bound with the purified preparation of pig kidney-Na+, K(+)-ATPase relative to active site of the enzyme was studied by EPR method. The number of Mn2+ ions in active site of the enzyme as well as that bound with lipids was determined from EPR spectra of paramagnetic manganese ions replacing magnesium ions were measured in frozen protein samples of Na2+, K(+)-ATPase at 77 K. It has been found that sulfhydryl group of the enzyme modified by PCMB-R and benzocarboline probe are placed at distances 38 A and 50 A, respectively, from Mn2+ ions in the active site of Na+, K(+)-ATPase. Evaluation of the immersion depth of the nitroxyl radical into protein globule showed that benzocarboline probe was immobilized near the macromolecular protein surface; there are two bound probe sites, distinguished by accessibility of ferricyanide ions.

Photochrome-labeling method in study of dynamics of biological systems.

The theoretical considerations and experimental evidences discussed in this paper indicate that quantitative study of photochromic processes in labeled objects open up new possibilities for investigating microviscosity and conformation transitions in biological systems. The proposed method of photochrome labeling features higher sensitivity and simplicity, and uses labels which are more stable under physiological conditions compared with traditional spin labels.

[Models for rotating spin labels and probes in proteins and membranes]. / Modeli vrashcheniia spinovykh metok i zondov v belkakh i membranakh.

A method of comparative analysis of ESR spectra has been proposed. It allows to distinguish between two models, slow reorientation of spin labels, and rapid rotation of the cone. Comparison of experimental data for a number of biological objects the theoretical predictions has shown that the rotation of nitroxyl fragment of spin labels can be described by the model of slow anisotropic rotation with correlation time 10(-6) less than or equal to tau less than or equal to 10(-8) s in conditions where the rotation of macromolecules is "frozen".

[Determination of depth of immersion of chlorophyll P680, pheophytin, and a secondary electron donor in the subchloroplast preparations of photosystem II from pea]. / Opredelenie glubiny pogruzheniia khlorofilla P680, feofitina i vtorichnogo donora élektrona v subkhloroplastnykh preparatakh fotosistemy II gorokha.

The immersion depths (r) of the main functional components of the reaction centres of the Photosystem 2 in the thylakoid membranes were determined by ESR at 77K. It was shown that P680(+), Pheo(-) (pheophytin), and Z(+) (secondary electron donor) the r value was 2-4, 4-7 and 14-20 A, respectively. On the basis of these and reference data a model of location of the Photosystem 2 reaction centre components in the photosynthetic membrane was suggested.

[Effect of hydration on the molecular dynamic properties of human serum albumin at low temperatures]. / Vliianie gidratatsii na molekuliarno-dinamicheskie svoistva syvorotochnogo al'bumina cheloveka v oblasti nizkikh temperatur.

The effect of temperature and hydration on phosphorescence of chromatophores and on saturation curves of ESR spectra of spin labels covalently bound to human serum albumin was studied. It has been shown that at 90-260 degrees K albumin hydration results in intensification of motions of hydrophobic parts with low frequencies (vc less than or equal to 10(3) s-1) and does not affect the motions of hydrophobic and surfacial parts with high frequency.

[Quantitative estimate of major factors determining the kinetics of electron transfer reactions with cytochrome c]. / Kolichestvennaia otsenka osnovnykh faktorov, opredeliaiushchikh kinetiku reaktsii élektronnogo perenosa s uchastiem tsitokhroma c.

Quantitative estimation of basic factors determining the electron transfer rate constant between cytochrome c and inorganic metal complexes and electron exchange rate constant based on the theory of nonadiabatic electron transfer in polar media is presented.

[Study of the effect of the microenvironment on magnetic resonance parameters of spin-labeled human serum albumin in a 2-mm ESR range]. / Izuchenie vliianiia mikrookruzheniia na magnitno-rezonansnye parametry spin-mechenogo syvorotochnogo al'bumina cheloveka v 2-mm diapazone.

Basic values of g-tensor and Azz component of HF tensor of two spin labels and spin probe on HSA and nitroxyl radicals HO-15, HO-34 in the solvents of different polarity were measured by 2 mm band ESR of 2 mm range. Magnetic-resonance parameters of liophylized and water-solved spin-labeled HSA were shown to correspond to the parameters of the solvents of the label HO-15 and HO-34 in ethyl alcohol and water. A conclusion was drawn concerning the identity of microenvironment of the nitroxyl fragment of liophylized HSA and frozen solution of the label HO-15 and HO-34 in ethyl alcohol and solvatation of the nitroxyl fragment of spin-labeled HSA and label HO-15 (HO-34) by water molecules.