EPR Spin Probe Research Articles

Bovine Serum Albumin Effect on Collapsing PNIPAM Chains in Aqueous Solutions: Spin Label and Spin Probe Study.

The influence of bovine serum albumin (BSA) on collapsing poly(N-isopropylacrylamide) (PNIPAM) chains was studied with turbidimetry and spin probe and spin label electron paramagnetic resonance spectroscopy. An increased ratio of collapsed chains in aqueous solutions in the narrow temperature region near the LCST appeared in the presence of 2.5-10 wt% BSA. The spin probe EPR data indicate that the inner cavities of the BSA dimers are probably responsive to the capture of small hydrophobic or amphiphilic molecules, such as TEMPO nitroxyl radical. The observed features of the structure and dynamics of inhomogeneities of aqueous PNIPAM-BSA solutions, including their mutual influence on the behavior of the polymer and protein below the LCST, should be considered when developing and investigating PNIPAM-based drug delivery systems.

A New Quantitative Method of Express Analysis of Natural Rubber Directly in the Root of the Rubber Plant by the Spin Probe EPR Method

A New Quantitative Method of Express Analysis of Natural Rubber Directly in the Root of the Rubber Plant by the Spin Probe EPR Method

Dependence of state transitions on illumination time in arabidopsis and barley plants.

Solar energy absorbed by plants can be redistributed between photosystems in the process termed "state transitions" (ST). ST represents a reversible transition of a part of the PSII light harvesting complex (L-LHCII) between photosystem II (PSII) and photosystem I (PSI) in response to the change in light spectral composition. The present work demonstrates a slower development of the state 1 to state 2 transition, i.e., L-LHCII transition from PSII to PSI, in the leaves of dicotyledonous arabidopsis (Arabidopsis thaliana) than in the leaves of monocotyledonous barley (Hordeum vulgare) plants that was assessed by the measurement of chlorophyll a fluorescence at 77 K and of chlorophyll a fluorescence at room temperature. It is known that the first step of the state 1 to state 2 transition is phosphorylation of Lhcb1 and Lhcb2 proteins; however, we detected no difference in the rate of accumulation of these phosphorylated proteins in the studied plants. Therefore, the parameters, which possibly affect the second step of this transition, i.e., the migration of L-LHCII complexes along the thylakoid membrane, were evaluated. Spin-probe EPR measurements demonstrated that the thylakoid membranes viscosity in arabidopsis was higher compared to that in barley. Moreover, confocal microscopy data evidenced the different size of chloroplasts in the leaves of the studied species being larger in arabidopsis. The obtained results suggest that the observed deference in the development of the state 1 to state 2 transition in arabidopsis and barley is caused by the slower L-LHCII migration rate in arabidopsis than in barley plants rather than by the difference in the Lhcb1 and Lhcb2 phosphorylation.

Comparative Study of Pd-Mayenite Catalysts Prepared via Aerogel Approaches.

Pd-containing catalysts based on highly dispersed aerogel-derived mayenite were prepared via two approaches. The Pd@C12A7 sample was obtained through the addition of Pd nitrate solution to a fresh Ca(OH)2-Al(OH)3 gel. Pd/C12A7 was synthesized through conventional wet impregnation of the aerogel-derived mayenite. The evolution of the textural characteristics of the support (C12A7) depending on the calcination temperature was investigated. Pd-containing samples were explored using transmission electron microscopy and spin probe EPR spectroscopy. Using the latter method, the presence of active oxygen species capable of producing nitroxyl radicals from diphenylamine was observed. The activity of these species and the reproducibility of their redox behavior were studied in three cycles of temperature-programmed reduction in both hydrogen and CO atmospheres. A prompt thermal aging technique was used to access and compare the activity of the samples towards CO oxidation. The state of Pd species before and after the aging procedure was studied via UV-Vis spectroscopy. It was found that the dispersion of PdO was higher in the case of the Pd/C12A7 catalysts compared to the Pd@C12A7 sample. This is why the Pd/C12A7 catalyst demonstrated higher activity in CO oxidation and better reducibility in TPR cycles.

Novel Orientation-Sensitive Spin Probes for Graphene Oxide Membranes Study.

Spin probe EPR spectroscopy is currently the only method to quantitatively report on the orientational ordering of graphene oxide membranes. This technique is based on the analysis of EPR spectra of a membrane containing stable radicals sorbed on oxidized graphene planes. The efficiency of the method depends on the spin probe structure; therefore, it is important to find stable paramagnetic substances that are most sensitive to the alignment of graphene oxide membranes. In the present work, three novel stable nitroxide radicals containing aromatic fragments with two nitrogen atoms were tested as spin probes to study graphene oxide membranes. The spin-Hamiltonian parameters of the radicals in graphite oxide powder and orientational order parameters of the probes inside graphene oxide membrane were determined. The sensitivity of one of these radicals to membrane orientational ordering was found to be higher than for any of spin probes used previously. A likely reason for this higher sensitivity is the presence of heteroatoms which can facilitate interaction between paramagnetic molecules and oxygen-containing groups on the inner surface of the membrane. The new high-sensitivity spin probe may significantly increase the potential of EPR spectroscopy for studying the internal structure of graphene oxide membranes.

Structure and Dynamics of Inhomogeneities in Aqueous Solutions of Graft Copolymers of N-Isopropylacrylamide with Lactide (P(NIPAM-graft-PLA)) by Spin Probe EPR Spectroscopy.

Coil-to-globule transition and dynamics of inhomogeneities in aqueous solutions of graft copolymers of NIPAM with different content of oligolactide groups were studied using spin probe continuous wave EPR spectroscopy. The technique of the suppressing of TEMPO as spin probe by spin exchange with Cu2+ ions was applied. This approach allowed us to detect individual EPR spectra of the probe in collapsed globules and estimate its magnetic and dynamic parameters reliably. The formation of inhomogeneities at temperatures lower than the volume phase transition temperature measured via transmission, and differential scanning calorimetry was fixed. An increase in oligolactide content in copolymers leads to the formation of looser globules, allowing for the exchange of the probe molecules between the globules and the external solution.

Interaction between meso-tetra-(4-hydroxyphenyl)porphyrin and SDS in aqueous solutions: Premicellar porphyrin-surfactant J-aggregate formation

Interactions between meso-tetra(4-hydroxyphenyl)porphyrin (THPP) and anionic sodium n-dodecyl sulfate (SDS) in a wide surfactant concentration range (0.5–50 mM) have been studied by UV–vis absorption and fluorescence spectroscopy, resonance light scattering (RLS), spin probe EPR spectroscopy and dynamic light scattering (DLS). J-type aggregate formation has been observed for THPP within 2–5 mM surfactant concentration range, accompanied by a significant bathochromic shift of the absorption bands, fluorescence quenching and emergence of an intense RLS signal. Porphyrin binding to the surfactant micelles above the cmc and aggregate formation below the cmc have been observed in SDS solutions containing THPP by DLS and EPR spectroscopy. SEM and optical microscopy revealed a rod-like morphology of the microparticles crystallized from an air dried suspension of THPP J-aggregates. The results obtained contribute to understanding of the mechanism of porphyrin-surfactant interactions both below and above the surfactant cmc.

Molecular dynamics and partition of impurity molecules in silicates during sol–gel processes: spin probe EPR study

Molecular dynamics and partition of impurity molecules in silicates during sol–gel processes: spin probe EPR study

Confinement dependence of protein-associated solvent dynamics around different classes of proteins, from the EPR spin probe perspective.

Protein function is modulated by coupled solvent fluctuations, subject to the degree of confinement from the surroundings. To identify universal features of the external confinement effect, the temperature dependence of the dynamics of protein-associated solvent over 200-265 K for proteins representative of different classes and sizes is characterized by using the rotational correlation time (detection bandwidth, 10-10-10-7 s) of the electron paramagnetic resonance (EPR, X-band) spin probe, TEMPOL, which is restricted to regions vicinal to protein in frozen aqueous solution. Weak (protein surrounded by aqueous-dimethylsulfoxide cryosolvent mesodomain) and strong (no added crysolvent) conditions of ice boundary confinement are imposed. The panel of soluble proteins represents large and small oligomeric (ethanolamine ammonia-lyase, 488 kDa; streptavidin, 52.8 kDa) and monomeric (myoglobin, 16.7 kDa) globular proteins, an intrinsically disordered protein (IDP, β-casein, 24.0 kDa), an unstructured peptide (protamine, 4.38 kDa) and a small peptide with partial backbone order (amyloid-β residues 1-16, 1.96 kDa). Expanded and condensate structures of β-casein and protamine are resolved by the spin probe under weak and strong confinement, respectively. At each confinement condition, the soluble globular proteins display common T-dependences of rotational correlation times and normalized weights, for two mobility components, protein-associated domain, PAD, and surrounding mesodomain. Strong confinement induces a detectable PAD component and emulation of globular protein T-dependence by the amyloid-β peptide. Confinement uniformly impacts soluble globular protein PAD dynamics, and is therefore a generic control parameter for modulation of soluble globular protein function.

Resolution and characterization of confinement- and temperature-dependent dynamics in solvent phases that surround proteins in frozen aqueous solution by using spin-probe EPR spectroscopy.

Spin probe electron paramagnetic resonance spectroscopy is applied to characterize the dynamics of concentric hydration and mesophase solvent domains that surround proteins within the ice boundary in frozen aqueous solutions. The solvent dynamics are tuned by variation of temperature (190-265K) and by the degree of ice boundary confinement, which is modulated by the volume of added cryosolvent (0-~50Å separation distance from protein surface). Goals are to: (1) characterize the protein-coupled solvent dynamics on correlation time scales of ~10-10<τ<10-7s, and spatial scales from protein surface to periphery of the surrounding solution, from the perspective of a free, small-molecule (~7Å diameter) probe, and (2) reveal properties of the solvent-protein coupling that can be correlated with protein functions, that are measureable under the same conditions. Rotational mobility of the nitroxide spin probe, TEMPOL, resolves and tracks two solvent components, the protein-associated domain (PAD; akin to hydration layer) and surrounding mesodomain, through their distinct temperature- and confinement-dependent values of τ and normalized weight. Detailed protocols are described for simulation of two-component nitroxide EPR spectra, which are categorized by line shape regime and guided by a library of template spectra and simulation parameters derived from two model soluble globular proteins. The order-disorder transition in the PAD, which is a universal feature of protein-coupled solvent dynamics, provides a well-defined, tunable property for elucidating mechanism in solvent-protein-function dynamical coupling. The low-temperature mesodomain system and EPR spin probe method are generally applicable to reveal solvent contributions to a broad range of macromolecule-mediated biological processes.

Hydrogen-Bonded Complexes of Neutral Nitroxide Radicals with 2-Propanol Studied by Multifrequency EPR/ENDOR

The hydrogen bond plays a key role in weak directional intermolecular interactions. It is operative in determining molecular conformation and aggregation, and controls the function of many chemical systems, ranging from inorganic, organic to biological molecules. Although an enormous amount of spectroscopic information has been collected about hydrogen-bond formation between molecules with closed-shell electronic configuration, the details of such interactions between open-shell radicals and closed-shell molecules are still rare. Here we report on an investigation of hydrogen-bonded complexes between pyrroline-type as well as piperidine-type neutral nitroxide radicals and an alcohol, i.e., 2-propanol. These nitroxide radicals are commonly used as EPR spin labels and probes. To obtain information on the geometry of the complexes and their electronic structure, multi-resonance EPR techniques at various microwave frequencies (X-, Q-, W-band, 244 GHz) have been employed in conjunction with DFT calculations. The planar five-membered ring system of the pyrroline-type nitroxide radical was found to form exclusively well-defined in-plane σ-type hydrogen-bonded complexes with one 2-propanol molecule in the first solvation shell in frozen solution. The measured hyperfine parameters of the hydrogen-bridge proton and the internal magnetic parameters describing the electron Zeeman and the electron-nuclear hyperfine and nuclear quadrupole interactions are in good agreement with values predicted by state-of-the-art DFT calculations. In contrast, multi-resonance EPR on the non-planar six-membered ring system of the piperidine-type nitroxide radical (TEMPOL) reveals a more complex situation, i.e., a mixture of a σ-type with, presumably, an out-of-plane π-type complex, both present in comparable fraction in frozen solution. For TEMPOL, the DFT calculations failed to predict magnetic interaction parameters that are in good agreement with experiment, apparently due to the considerable flexibility of the nitroxide and hydrogen-bonded complex. The detailed information about nitroxide/solvent complexes is of particular importance for Dynamic Nuclear Polarization (DNP) and site-directed spin-labeling EPR studies that employ nitroxides as polarizing agents or spin labels, respectively.

Low-temperature molecular motions in a deep eutectic solvent choline chloride/urea studied by spin-probe EPR

Low-temperature molecular motions in a deep eutectic solvent choline chloride/urea studied by spin-probe EPR

Versatile Para-Substituted Pyridine Lanthanide Coordination Complexes Allow Late Stage Tailoring of Complex Function.

A series of cationic and neutral p-Br and p-NO2 pyridine substituted Eu(III) and Gd(III) coordination complexes serve as versatile synthetic intermediates. Nucleophilic aromatic substitution occurs readily at the para position under mild conditions, allowing C-N and C-C bond forming reactions to take place, permitting the introduction of azide, amino and alkynyl substituents. For Eu(III) complexes, this approach allows late stage tuning of absorption and emission spectral properties, exemplified by the lowering of the energy of an LMCT transition accompanied by a reduction in the Eu-Npy bond length. Additionally, these complexes provide direct access to the corresponding Eu(II) analogues. With the Gd(III) series, the nature of the p-substituent does not significantly change the EPR properties (linewidth, relaxation times), as required for their development as EPR spin probes that can be readily conjugated to biomolecules under mild conditions.

Bee Venom Melittin Disintegrates the Respiration of Mitochondria in Healthy Cells and Lymphoblasts, and Induces the Formation of Non-Bilayer Structures in Model Inner Mitochondrial Membranes.

In this paper, we examined the effects of melittin, a bee venom membrane-active peptide, on mitochondrial respiration and cell viability of healthy human lymphocytes (HHL) and Jurkat cells, as well as on lymphoblasts from acute human T cell leukemia. The viability of melittin-treated cells was related to changes in O2 consumption and in the respiratory control index (RCI) of mitochondria isolated from melittin-pretreated cells as well as of mitochondria first isolated from cells and then directly treated with melittin. It was shown that melittin is three times more cytotoxic to Jurkat cells than to HHL, but O2 consumption and RCI values of mitochondria from both cell types were equally affected by melittin when melittin was directly added to mitochondria. To elucidate the molecular mechanism of melittin’s cytotoxicity to healthy and cancer cells, the effects of melittin on lipid-packing and on the dynamics in model plasma membranes of healthy and cancer cells, as well as of the inner mitochondrial membrane, were studied by EPR spin probes. The affinity of melittin binding to phosphatidylcholine, phosphatidylserine, phosphatidic acid and cardiolipin, and binding sites of phospholipids on the surface of melittin were studied by 31P-NMR, native PAGE and AutoDock modeling. It is suggested that the melittin-induced decline of mitochondrial bioenergetics contributes primarily to cell death; the higher cytotoxicity of melittin to cancer cells is attributed to its increased permeability through the plasma membrane.

Mobility of liquids intercalated into the interplane space of graphite oxide as revealed by a combination of 19F NMR, 1H NMR and EPR spin probe methods.

A combination of 19F and 1H NMR with the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence was used to examine the mobility of liquids in the interplane space of graphite oxide (GO) for the first time. The proposed approach allows for the reduction of NMR signals from immobile hydrogen-containing groups on the surface of GO and for monitoring of the molecular mobility of intercalated liquids. The mobile fractions of H2O, CH3CN and CF3CH2OH were detected inside the corresponding swollen GO samples. For H2O, the amount of mobile liquid showed a peculiar temperature dependence where a certain portion survived well below 273 K. The sensitivity of the proposed 1H NMR + CPMG procedure is also compared to the sensitivity of the EPR nitroxide spin probe method.

Dielectric and Electrostatic Properties of the Silica Nanoparticle–Water Interface by EPR of pH-Sensitive Spin Probes

Interfacial electrostatic properties of monodisperse silica nanoparticles (SiNPs) in aqueous suspensions as a function of bulk pH were characterized by spin labeling EPR of two ionizable nitroxides...

Correction of Cold Damage to Mammalian Erythrocytes by Chlorpromazine to Influence the Dynamic Structure of a Membrane

Abstract—The resistance of mammalian erythrocytes (human, dog, horse, bull, and rabbit) to cold shock in the presence of chlorpromazine was studied; its effect on the state of erythrocyte membranes was investigated. The bond strength between membrane phospholipids of mammalian erythrocytes and the characteristics of the cold shock of these cells was determined with the correlation analysis. It has been established that erythrocytes of mammals whose membranes contain a high content of sphingomyelin and phosphatidylethanolamine and a low content of phosphatidylcholines are more resistant to cold shock. Chlorpromazine showed high efficiency in protecting mammalian erythrocytes against cold shock. The dynamic structure of membranes of human erythrocytes treated with the chlorpromazine was studied by EPR of spin-probes. The use of a set of spin-labeled probes: palmitic acid amide, 5-DOXYL stearic acid, and 16-DOXYL stearic acid, which allow studying the micro-viscosity parameters of the lipid bilayer of erythrocyte membranes in the transmembrane direction, made it possible to establish the locations of chlorpromazine action, namely, in the region of polar heads and hydrophobic tails of membrane phospholipids.

Особенности применения ароматических молекул-зондов для ЭПР -спектроскопии в исследовании кислотных свойств поверхности модифицированных алюмооксидных каталитических систем

В обзоре систематизированы работы, посвящённые применению метода спектроскопии ЭПР в варианте ароматических молекул-зондов для исследования гетерогенных алюмооксидных катализаторов, а также дополнены новыми результатами исследований, проведённых в ИППУ СО РАН. Рассмотрены перспективы метода и представления о механизме взаимодействия молекул-зондов с активными центрами поверхности и их природе. Отмечено наличие противоречий во взглядах на механизмы, а также что современная точка зрения склоняется к участию БКЦ в ионизации ароматических молекул-зондов.

Effect of Zinc–Porphyrin Complex on the Structure and Properties of Poly(3-hydroxybutyrate) Ultrathin Fibers

The effect of small amounts of the zinc–tetraphenylporpyrin complex (1–5%) on the structure and properties of ultrathin fibers based on poly(3-hydroxybutyrate) is investigated using differential scanning calorimetry, X-ray diffraction, spin-probe EPR spectroscopy, scanning electron microscopy, and IR spectroscopy. It is shown that, upon introduction of the porphyrin complex in the fibers, the fraction of paracrystalline structures decreases and molecular mobility in the amorphous regions of the polymer slows down, while the degree of crystallinity remains unchanged. Annealing of the fibers at 140°C leads to an increase in the content of paracrystalline structures and in the degree of crystallinity of the fibers.

Features of the Interaction between Dodecyltrimethylammonium Bromide Micelles and Sodium 4-Styrene Sulfonate

Spin probe EPR spectroscopy and UV spectroscopy have been employed to study the interaction between a hydrophobic anionic monomer, sodium styrene sulfonate, and micelles of a cationic surfactant, dodecyltrimethylammonium bromide. It has been shown that the interaction with the monomer decreases the critical micelle concentration of the surfactant in aqueous solutions. It has been found that the monomer is incorporated into micelles; therefore, the presence of the surfactant may hinder polymerization of the monomer.