[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.

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.

Novel fluorescence method for real-time monitoring of nitric oxide dynamics in nanoscale concentration.

A novel assay was developed for the measurement of nitric oxide. The proposed method is based on fluorescence, using a fluorophore-heme dual functionality probe (FHP). The heme group can serve as an effective NO-trap, due to its very fast reaction with NO and the high stability of the resulting complex.

The novel FluoroChrome ImmunoAssay -- (FCIA). The role of molecular environment upon molecular structure exemplified by constriction of a flourescence-photochrome flanked by two proteins.

A rapid, sensitive, and quantitative novel immunoassay [FluoroChrome ImmunoAssay, FCIA] technique was developed which auspiciously combines both the high sensitivity of fluorescence measurements with the high specificity of an antibody. As opposed to existing immunoassays, FCIA is performed without separation of antibody-bound haptens from those that are free, and utilizes fluorescence measurements from widely available standard commercial fluorimeters. FCIA is based on the hypothesis that an appropriately designed stilbene-antigen analogue probe will suffer considerable steric hindrance to trans-cis photoisomerization when bound within the combined constraints of both an antibody binding site and a second globular protein.

Neonatal blood is more resistant to oxidative stress induced by stable nitroxide radicals than adult blood.

Neonate erythrocytes are more susceptible to oxidizing drugs than adults; however, there are controversial reports in the literature regarding the total antioxidant capacity of neonate blood. Stable nitroxide radicals (NRs) are reduced by blood and some other biological materials to the corresponding hydroxylamines. The kinetics of the nitroxide's disappearance using electron paramagnetic resonance (EPR) spectroscopy, provides useful biochemical and biophysical information about the antioxidant properties of biological systems.

Structural features of transiently modified beta-lactoglobulin relevant to the stable binding of large hydrophobic molecules.

Binding sites for hydrophobic molecules on bovine beta-lactoglobulin, and their susceptibility to temperature, were studied by using various spectroscopic probes. Binding of probes carrying a single fluorophore moiety, a single nitroxide moiety, or both moieties on the same molecule, was followed by EPR and fluorescence. The presence of a fatty acid side chain in the dual probes was found to be required for binding to beta-lactoglobulin.

Study of rare encounters in a membrane using quenching of cascade reaction between triplet and photochrome probes with nitroxide radicals.

Measurements of active encounters between molecules in native membranes containing ingredients, including proteins, are of prime importance. To estimate rare encounters in a high range of rate constants (rate coefficients) and distances between interacting molecules in membranes, a cascade of photochemical reactions for molecules diffusing in multilamellar liposomes was investigated. The sensitised cascade triplet cis-trans photoisomerisation of the excited stilbene involves the use of a triplet sensitiser (Erythrosin B), a photochrome stilbene-derivative probe (4-dimethylamino-4'-aminostilbene) exhibiting the phenomenon of trans-cis photoisomerisation, and nitroxide radicals (5-doxyl stearic acid) to quench the excited triplet state of the sensitiser.

Detection of nitric oxide from pig trachea by a fluorescence method.

A nitronyl nitroxide radical covalently linked to an organic fluorophore, pyrene, was used to detect nitric oxide (NO) from freshly excited tissues. This approach is based on the phenomenon of the intramolecular fluorescence quenching of the fluorophore fragment by the nitroxide. The pyrene-nitronyl (PN) reacts with NO to yield a pyrene-imino nitroxide radical (PI) and NO(2).

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*.

Molecular dynamics investigation of an antibody binding site by the fluorescence-photochrome method.

A combined fluorescence-photochrome approach was used for investigation of the molecular dynamics antiDNP antibody binding site and its cavity. A 4-(N-2,4-dinitrophenylamino)-4'-(N,N'-dimethylamino)stilbene (StDNP) fluorescence DNP analog was incorporated into the antibody binding site. This was followed by measurements of fluorescence and photochrome parameters such as the StDNP excitation and emission spectra, fluorescence lifetime, steady-state and time-resolved fluorescence polarization, kinetics of trans-cis and cis-trans photoisomerization, and fluorescence quenching by nitroxide radicals freely diffused in solution.

Heterogeneity in the structural dynamics of Sulfolobus solfataricus beta-glycosidase revealed by electron paramagnetic resonance and frequency domain fluorometry.

The local and global dynamics of the Sulfolobus solfataricus beta-glycosidase were studied by electron spin resonance and time-resolved fluorescence techniques. For electron paramagnetic resonance (EPR) investigations, the protein was covalently modified by the maleimido nitroxide spin label, which is specific for cysteine -SH groups, at position 344 and at position 101, where Ser-101 was changed into a cysteine by site-directed mutagenesis. The greater reactivity of exposed Cys-101 suggested the exclusive modification of this amino acid compared with Cys-344.

Effect of ionic strength on the binding of ascorbate to albumin.

A fluorophore-nitroxide free radical dual-functional probe (FN) was utilized to study the kinetics of ascorbate (AH(-)) binding to Bovine Serum Albumin (BSA). Since the free radical fragment in the FN probe intramolecularly quenches fluorescence, ascorbate reduction of the nitroxide function is accompanied by a concomitant fluorescence intensity increase from the fluorophore. Thus, both fluorescence and the EPR techniques could be utilized to measure the reaction rate.

A fluorescent-photochrome method for the quantitative characterization of solid phase antibody orientation.

A fluorescent-photochrome method of quantifying the orientation and surface density of solid phase antibodies is described. The method is based on measurements of quenching and rates of cis-trans photoisomerization and photodestruction of a stilbene-labeled hapten by a quencher in solution. These experimental parameters enable a quantitative description of the order of binding sites of antibodies immobilized on a surface and can be used to characterize the microviscosity and steric hindrance in the vicinity of the binding site.

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).

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.

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.

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.

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.

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.

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.

[Model molybdenum-sulfur and molybdenum-iron-sulfur protein complexes].

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.

[Statistical analysis of electron microphotographs of bioobjects, labelled by electron-dense mercarbide markers].

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.

[Interaction of spin labels and probes with Na+,K+-ATPase].

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.

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.