Spin Probe Method Research Articles

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in Russian

ESR Study of the Nature of Melanin from Micellar Fungi Nadsoniella Nigra

Melanin-containing powders and their aqueous solutions have been studied by electron spin resonance (ESR). Lorentzian lineshape, temperature dependence of magnetic susceptibility and linewidth resemble those for conducting polymers in which polyconjugated chemical links between their paramagnetic centers play an important role. Aqueous solution of one powder has been studied by the spin-probe method. It has been found that the addition of probes Fe(CN) 6 3− does not affect substantially both the width and the amplitude of melanin ESR lines probably due to strong electrostatic repulsion between the probes and negatively charged surrounding of melanin. The addition of probes Co2+ decreases the amplitude of melanin ESR line and does not affect its width. This may be explained by adsorption of the probes on negatively charged sites in the vicinity of melanin paramagnetic centers.

Оценка влияния углеродных нанохорнов на микровязкость мембран эритроцитов и белки плазмы крови крыс методом спиновых зондов

Оценка влияния углеродных нанохорнов на микровязкость мембран эритроцитов и белки плазмы крови крыс методом спиновых зондов

The study of o/w emulsions using the rotating viscometer method and the method of spin probes

The study of o/w emulsions using the rotating viscometer method and the method of spin probesThe rheological properties of o/w emulsions used as vehicles for semi-solid preparations (SSP) depend on the composition of o/w and w/o emulsifiers to a large extent, and it is related to the structure of their aggregates in emulsions. In order to control the rheological parameters of emulsions it is necessary to know the regularities of the influence of the composition and properties of emulsifiers on these parameters, as well as the mechanism of emulsion stabilization, which depends on the structure of the aggregates formed by emulsifiers.Aim. To study the relationship between the structure of the aggregates formed by emulsifiers and the rheological properties of o/w emulsions.Materials and methods. The objects of the study were o/w emulsions stabilized with macrogol-40 stearate (M40S), glyceryl monostearate 40-55 (type II) (GMS) and cetostearyl alcohol (CSA). The rheological studies were performed by the rotational viscometer method. The structure of aggregates of emulsifiers was studied by the spin probes method. Probes simulating o/w and w/o emulsifiers with different localization of the radical were used.Results and discussion. The apparent viscosity of emulsions stabilized with the o/w emulsifier (M40S) and a mixture of w/o emulsifiers (GMS and CSA) is maximal at a certain ratio between o/w and w/o emulsifiers. The increase in the rheological parameters correlates with the increase in the packing density of emulsifier molecules in the polar part of their aggregates; it in this case practically does not change at the level of the 5 and 16 carbon atoms of alkyl chains. This indicates formation of non-spherical aggregates which form a coagulation structure in the emulsion.Conclusions. The change in the ratio between o/w and w/o emulsifiers results in the change in the structure of their aggregates, and it affects the rheological properties of o/w emulsions. Based on the results of the studies it is possible to reasonably control the rheological parameters of preparations with emulsions as a vehicle.

The study of steroid distribution in emulsions by the spin probe method

The solubility of the active substance, which determines its dispersed state in a dosage form, is a significant pharmaceutical factor for providing the required functional characteristics of a medicinal product, its effectiveness, safety and, if necessary, bioavailability.Aim. To study the behavior of spin probes based on steroids in some solvents, micelles of nonionic surfactants, as well as emulsions both of type 1 and type 2 with a different composition of excipients in order to predict bioavailability, efficacy and safety of medicinal products with steroids.Materials and methods. The studies were carried out by the spin probe method. Two spin probes based on steroids with different localization of the nitroxide radical in their molecules were used.Results. The disperse state and localization of steroid molecules in some solvents, surfactant micelles and emulsions both of type 1 and type 2 were identified using the shape of EPR spectra, the isotropic constant values and the rotational correlation time of two different spin probes. It has been found out that the spin probes based on steroids with different hydrophilic-lipophilic properties are distributed differently in the dispersion medium and in the dispersed phase of emulsions. The emulsion type, the chemical nature, the composition and polarity of the dispersion medium and the dispersed phase also cause differences in distribution of steroids in emulsions.Conclusions. Different localization and disperse state of steroids in vehicles can affect the effectiveness of their pharmacological action, bioavailability in different routes of administration and the safety of drugs. In the pharmaceutical development of drugs it is necessary to study the solubility of steroids, their distribution between the dispersed phase and the dispersion medium of emulsions, the disperse state in the vehicles, as well as to carry out biopharmaceutical and/or pharmacological screening studies in order to select the optimal composition of the medicinal product.

Оценка взаимодействия окисленного графена с мембранами эритроцитов и белками плазмы крови крыс методом спиновых зондов

Оценка взаимодействия окисленного графена с мембранами эритроцитов и белками плазмы крови крыс методом спиновых зондов

Effect of Solution Ionic Strength on the pKa of the Nitroxide pH EPR Probe 2,2,3,4,5,5-Hexamethylimidazolidin-1-oxyl.

Spin probe and spin labeling Electron Paramagnetic Resonance methods are indispensable research tools for solving a wide range of bioanalytical problems-from measuring microviscosity and polarity of phase-separated liquids to oxygen concentrations in tissues. One of the emerging uses of spin probes are the studies of proton transfer-related and surface electrostatic phenomena. The latter Electron Paramagnetic Resonance methods rely on molecular probes containing an additional functionality capable of reversible ionization (protonation, in particular) in the immediate proximity to an Electron Paramagnetic Resonance-active reporter group, such as (N-O•) for nitroxides. The consequent formation of protonated and nonprotonated nitroxide species with different magnetic parameters (A iso, g iso) could be readily distinguished by Electron Paramagnetic Resonance. Bioanalytical Electron Paramagnetic Resonance studies employing pH-sensitive paramagnetic probes typically involve determination of the equilibrium constant (pK a) between the protonated and nonprotonated forms of the nitroxide. However, any chemical equilibrium involving charged species, such as ionization of acids and bases, and so the reversible protonation of the nitroxide, is known to be affected by an ionic strength of the solution. Currently, only scarce data for the effect of the solution ionic strength on the experimental pK a's of the ionizable nitroxides can be found in the literature. Here we have carried out a series of Electron Paramagnetic Resonance titration experiments for aqueous solutions of 2,2,3,4,5,5-hexamethylimidazolidin-1-oxyl (HMI) nitroxide known for one of the largest differences in the isotropic nitrogen hyperfine coupling constant A iso between the protonated and nonprotonated forms. Electrolyte concentration was varied over an exceptionally large range (i.e., from 0.05 to 5.0 M) to elucidate the effect of ionic strength on the ionization constant of this pH-sensitive Electron Paramagnetic Resonance probe and the data were compared to the Debye-Hückel limiting law. Effects of the ionic strength on the magnetic parameters of the ionizable nitroxides are also discussed.

Impregnation of polymers with 2,2,6,6-tetramethyl-4-oxo-piperidine-1-oxyl (TEMPONE) paramagnetic probe in sub- and supercritical CO2

The spin probe method is used to study the impregnation of polycarbonate (PC) based on bisphenol A, polyethylene oxide (PEO), and crosslinked acrylamide–acrylic acid copolymer (PAA) with organic molecules in sub- and supercritical CO2 media. Electron spin resonance (EPR) data show that, at 196 bar and 307 K, 2,2,6,6-tetramethyl-4-oxo-piperidine-1-oxyl (TEMPONE) paramagnetic spin probe molecules penetrate into the PC and PEO matrices, which are, respectively, in the glassy and elastic states under normal conditions. The degree of impregnation of PAA under these conditions is negligibly small. Estimates of the local concentration of probe molecules show that, in the PEO matrix, TEMPONE is distributed much more uniformly than in the PC matrix. Analysis of the effect of temperature on the shape of the EPR spectra of the radical in the polymer matrix shows that, under the same conditions, the mobility of TEMPONE molecules in the PEO matrix is much higher than in the PC matrix. The results suggest that the spin probe method is promising for studying the characteristics of macro- and micro-processes in polymer–supercritical fluid solvent–organic molecule ternary systems.

CO Oxidation over Pd/ZrO₂ Catalysts: Role of Support's Donor Sites.

A series of supported Pd/ZrO2 catalysts with Pd loading from 0.2 to 2 wt % was synthesized. The ZrO2 material prepared by a similar technique was used as a reference sample. The samples have been characterized by means of transmission electron microscopy (TEM), X-ray diffraction analysis (XRD), X-ray photoelectron spectroscopy (XPS), temperature-programmed reduction (TPR), testing reaction of ethane hydrogenolysis (HGE), N2 adsorption, and electron paramagnetic resonance (EPR) spectroscopy. 1,3,5-trinitrobenzene was used as a probe molecule for the EPR spin probe method. The catalytic performance of samples was tested in the model reaction of CO oxidation. It was shown that the concentration of donor sites of support measured by EPR spin probe correlates with catalytic behavior during light-off tests. Low concentration of donor sites on a support’s surface was found to be caused by the presence of the specific surface defects that are related to existence of coordinately unsaturated structures.

Molecular mobility of nanocellulose hydrogels

The molecular mobility of nanocellulose hydrogels isolated from microcrystalline cellulose is evaluated using the spin probe method, from the correlation time τ (s) and rotational frequency ν = 1/τ(s–1) of stable nitroxyl radicals introduced into the medium under study. In an aqueous gel medium, the EPR spectrum of the probe features an anisotropic triplet of frozen particles over a temperature range of 77 to 265 K. In an aqueous–ethanolic gel solution, the temperature of onset of rotation of the radical is 85 K lower. The rotational correlation time is determined from the parameters of the EPR spectrum recorded in the temperature range of 180–290 K. The resulting Arrhenius temperature dependence logν = f(1/T) is used to evaluate the activation energy of rotation E of the radical and the preexponential factor ν0(s–1), the frequency of rotational vibrations of the particle around the equilibrium position. For the aqueous medium, E = 11.2 kcal/mol; in the presence of ethanol, E = 5.2 kcal/mol; the preexponential factors for the aqueous and aqueous–ethanolic media are ν0 = 7 × 1018 and 6 × 1014 s–1, respectively. The parameters E and ν0 measured in the pure solvents and in the samples containing nanocellulose differ little, which is indicative of a high hydrophobicity of the probe molecule (and hydrogel particles) and of their weak interaction with the environment. The high value (~1018 s–1) of the preexponential factor is explained in terms of the compensation effect of water.

Mobility of the spin probe TEMPO in glassy metathesis polymers with substituents containing flexible Si-O-Si groups

Glassy metathesis polymers (glass-transition temperatures of 110–236°C) containing substituents with flexible Si-O-Si groups are studied by the spin-probe method with the use of the stable nitroxyl radical TEMPO. A high mobility of the spin probe not correlating with the free volume of polymers is reported. These results and the earlier discovered thermodynamic selectivity of the indicated polymers (high permeability coefficients of higher hydrocarbons) make it possible to infer that Si-O-Si groups are responsible for the observed behavior similarly to that in siloxane rubbers.

Оценка влияния углеродных нанотрубок на микровязкость мембран эритроцитов методом спиновых зондов

Оценка влияния углеродных нанотрубок на микровязкость мембран эритроцитов методом спиновых зондов

Properties of aqueous-saline human placental extracts and their fractions after storage of placenta at various subzero temperatures

Objective: In this study we analyzed the influence of low temperature storage of placental tissue on the effect of obtained aqueous-saline placental extracts and their extract fractions on the state of membranes and cytosol of erythrocytes. Methods: Fractions of human placenta extract (HPE) were obtained with gel-chromatography method. Spontaneous hemolysis of erythrocytes was measured spectrophotometrically by free hemoglobin content in supernatant at 543 nm wavelength. Osmotic fragility (resistance) of erythrocytes was assessed by the level of hemolysis in 0.45% NaCl solution. Low-pH resistance of erythrocytes was evaluated by the time of 50% hemolysis in sodium-phosphate-citrate buffer (pH 3.8), calculating the data from kinetic curve of the change of optical density at 700 nm. Electron paramagnetic resonance (EPR) spin probe method was applied for analysis of cytosol dynamic state. Results: We recorded that low temperature storage of placental tissue allows decreasing membranotropic effect, which was inherent to certain aqueous-saline placental extracts and some of their fractions. At the same time, storage of placental tissue at both 20 С and 196 С does not influence the ability of HPE to decrease erythrocyte osmotic fragility. Erythrocytes exposed with HPE derived from placenta stored for 6 months at 20 С displayed decrease of low-pH resistance. This effect was inherent not only to the whole placental extract, but also to its certain fractions, especially those with molecular weight below 5 kDa. Conclusions: Storage of placental tissue at 196 С seems to be advantageous for preservation of studied properties of placental aqueous-saline extract in comparison to storage at 20 С.

Adsorption and molecular dynamics of low-molecular substances on modified montmorillonite nanoparticles

The effect of the structure of stable nitroxyl radicals on their adsorption from organic solvents on montmorillonite modified with cationic surfactants is examined. The influence of the nature and content of the modifier and solvent, as well as additional modification with cationic dyes, on the adsorption is considered. The kinetic parameters of the adsorption of decane, toluene, and ethanol from the gas phase are determined. The spin probe method is used to determine the phase state and distribution of adsorbed additives, as well as the influence of an additional adsorption of hydrocarbons on the molecular dynamics at the surface of nanoparticles of montmorillonite and nanopigments based on cationic dyes.

Electron paramagnetic resonance (EPR) investigation of skin structure of Psoriasis vulgaris

E paramagnetic resonance (EPR) spin-probe method was used to investigate structural aspects of psoriasis vulgaris stratum corneum (pv-SC). EPR spectra of the SC samples were analyzed using order parameter (S0) and rotational diffusion rates. A little, broad three-line pattern of 5-doxylstearic acid (5-DSA) in pv-SC was observed. The spectral pattern of pv-SC is quite different from those of control SC reported.The S0-values obtained for the pv-SC and the control were approximately 0.20 and 0.49, respectively. The statistical analysis suggests that the 0.20 value of pv-SC is significantly smaller than the 0.49 value of the control (p<0.01). The rotational diffusion rates for the probe motion in the SC were faster than those of the control. Moreover, there was no significant spectral difference of the glass-plate with the SC against the static magnetic field, except for the signal intensity.The present results suggest that the pv-SC is less rigid of the structure than that of the control SC, indicating irregular architecture of pv-SC.

Stratum Corneum Structure of Psoriasis Vulgaris Investigated by EPR Spin-Probe Method

Electron paramagnetic resonance (EPR) spin-probe method was used to investigate structural aspects of psoriasis vulgaris SC (pv-SC). EPR spectra of the SC samples were analyzed using order parameter (S 0) and rotational diffusion rates. A little, broad three-line pattern of 5-doxylstearic acid (spin probe) in pv-SC was observed. The spectral pattern of pv-SC is quite different from those of control SC reported. The S 0 values obtained for the pv-SC and the control were approximately 0.20 and 0.49, respectively. The statistical analysis suggests that the 0.20 value of pv-SC is significantly smaller than the 0.49 value of the control (p < 0.01). The rotational diffusion rates for the probe motion in the SC were faster than those of the control. Moreover, there was no spectral difference of the glass plate with the SC against the static magnetic field, except for the signal intensity. Therefore, the pv-SC is less rigid of the structure than that of the control SC, indicating irregular architecture of pv-SC. The present results suggest that the EPR assay is of great use for evaluating various SC function and can be extended to other skin diseases with abnormal keratinization.

Preparation and dynamic properties of an anisotropic natural rubber film as viewed by electron spin resonance–spin probe method

Natural rubber (NR) films with the thickness of about 1 mm were prepared by removing the liquid phase from NR latex, which was previously irradiated. The primary radiation dose varied from 0 kGy (for unirradiated NR) to 200 kGy. Dry NR films were uniaxially stretched, and the degree of deformation, defined as λ = l/l0 (l0 and l being the lengths of relaxed and uniaxially deformed sample, respectively), was varied from λ = 1.0 (relaxed state) to λ = 2.7. Samples were then irradiated with secondary dose, which was chosen to be 100 or 200 kGy. NR films were characterized by differential scanning calorimetry and electron spin resonance–spin probe method. In addition, samples were exposed to accelerated thermal aging, and changes in molecular dynamics and structure were obtained. It has been shown that the application of deformation during the cross‐linking leads to the restriction in segmental mobility for the lower secondary dose, whereas for the higher secondary dose, larger values of λ induce an opposite effect originating from the structural changes. Thermally aged samples show higher amount of the gel phase and consequently higher fraction of slow motional chain segments. POLYM. ENG. SCI., 53:2284–2291, 2013. © 2013 Society of Plastics Engineers

Influence of the temperature of calcination of bayerite-containing aluminum hydroxide pellets on the water vapor adsorption capacity and acid-base properties of alumina

The paper considers the influence of the temperature of calcination (350–550°C) of alumina-based water adsorbents prepared from bayerite-containing aluminum hydroxide on the static water sorption capacity at high and low relative air humidities and on the acid-base properties of the surface. The acid-base properties of aluminas have been determined by means of IR spectroscopy of adsorbed CO probe molecule (Lewis sites) and by the spin probe method, which was used to determine the concentrations of electron acceptor and electron donor sites. It has been demonstrated that, at a high relative air humidity (60%), there is no correlation between the static capacity of the drier and its acid-base properties, while at low relative humidities (1–1.5%), an increase in the concentration of electron donor sites and a decrease in the concentration of electron donor and Lewis acid sites enhance the water sorption capacity of alumina.

Noninvasive mapping of the redox status in septic mouse by in vivo electron paramagnetic resonance imaging

Increased reactive oxygen species (ROS) contribute to numerous brain disorders, and ROS generation has been examined in diverse experimental models of lipopolysaccharide (LPS)-induced inflammation. The in vivo electron paramagnetic resonance (EPR)/nitroxide spin probe method has been used to analyze the redox status in animal models modulated by ROS generation. In this study, a blood–brain barrier (BBB)-permeable nitroxide spin probe, 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (HMP), was used as a redox-sensitive nitroxide probe. Magnetic resonance images of mouse head after the injection of HMP showed that HMP was distributed throughout all regions of the mouse head including the brain, suggesting that HMP can reveal redox information in all regions of the mouse head. After the injection of HMP through the mouse tail vein 6 h after the injection of LPS, three-dimensional (3D) EPR images were obtained each minute under a field scanning of 0.3 s and with 81 projections. The reduction reaction of HMP in septic mouse heads was remarkably accelerated compared to that in control mice, and this accelerated reaction was inhibited by aminoguanidine and allopurinol, which inhibit enzymatic activities of induced nitric oxide synthase and xanthine oxidase, respectively. Based on the pharmacokinetics of HMP in mouse heads, the half-life mapping of HMP was performed in LPS-treated mouse head. Half-life maps clearly show a difference in the redox status induced by ROS generation in the presence or absence of inhibitors of ROS-generating enzymes. The present results suggest that a 3D in vivo EPR imaging system combined with BBB-permeable HMP is a useful noninvasive tool for assessing changes in the redox status in rodent models of brain disease under oxidative stress.

Detection and Imaging of Superoxide in Roots by an Electron Spin Resonance Spin-Probe Method

The detection, quantification, and imaging of short-lived reactive oxygen species, such as superoxide, in live biological specimens have always been challenging and controversial. Fluorescence-based methods are nonspecific, and electron spin resonance (ESR) spin-trapping methods require high probe concentrations and lack the capability for sufficient image resolution. In this work, a novel (to our knowledge), sensitive, small ESR imaging resonator was used together with a stable spin probe that specifically reacts with superoxide with a high reaction rate constant. This ESR spin-probe-based methodology was used to examine superoxide generated in a plant root as a result of an apical leaf injury. The results show that the spin probe rapidly permeated the plant's extracellular space. Upon injury of the plant tissue, superoxide was produced and the ESR signal decreased rapidly in the injured parts as well as in the distal part of the root. This is attributed to superoxide production and thus provides a means of quantifying the level of superoxide in the plant. The spin probe's narrow single-line ESR spectrum, together with the sensitive imaging resonator, facilitates the quantitative measurement of superoxide in small biological samples, such as the plant's root, as well as one-dimensional imaging along the length of the root. This type of methodology can be used to resolve many questions involving the production of apoplastic superoxide in plant biology.