Mobility Of Spin Probes Research Articles

A Combined Atomic and Molecular Probe Characterization of Aromatic Hydrocarbons via PALS and ESR: Methylbenzene.

A combined study of one of the simplest aromatic hydrocarbons, i.e., methylbenzene (toluene) (TOL), via the annihilation of an ortho-positronium (o-Ps) probe via positron annihilation lifetime spectroscopy (PALS) and the rotation dynamics of nitroxide spin probe 2,2,6,6-tetramethyl-piperidinyl-1-oxy (TEMPO) using electron spin resonance (ESR) over a wide temperature range, 10–300 K, is reported. The o-Ps lifetime, τ3, and the relative o-Ps intensity, I3, as a function of temperature exhibit changes defining several characteristic PALS temperatures in the slowly and rapidly cooled samples. Similarly, the spectral parameter of TEMPO mobility in TOL, 2Azz‘, and its correlation time, τc, reveal several effects at a set of the characteristic ESR temperatures, which were determined and compared with the PALS results. Finally, the physical origins of the changes in free volume expansion and spin probe mobility are revealed. They are reflected in a series of the mutual coincidences between the characteristic PALS and ESR temperatures and appropriate complementary thermodynamic and dynamic techniques.

Influence of the Electric Charge of Spin Probes on Their Diffusion in Room-Temperature Ionic Liquids.

The rotational and translational diffusion of negatively charged and uncharged spin probes in five imidazolium-based room-temperature ionic liquids (RTILs), 1-ethyl-3-methylimidazolium tetrafluoroborate, emimBF4, 1-butyl-3-methylimidazolium tetrafluoroborate, bmimBF4, 1-octyl-3-methylimidazolium tetrafluoroborate, omimBF4, 1-octyl-3-methylimidazolium hexafluorophosphate, omimPF6, and 1-octyl-3-methylimidazolium chloride, omimCl, has been studied by means of electron paramagnetic resonance spectroscopy. Detailed analyses of the spin-Hamiltonian parameters and spin exchange interactions have been carried out. The temperature dependences of the line broadening induced by the electronic dipole-dipole interaction and the electron spin exchange coupling are determined. The translational mobility of spin probes is semiquantitatively characterized and successfully explained in the framework of a hypothesis based on the assumption of polar and unpolar domains within the RTILs.

Coupling of ethanolamine ammonia-lyase protein and solvent dynamics characterized by the temperature-dependence of EPR spin probe mobility and dielectric permittivity.

Electron paramagnetic resonance (EPR) spectroscopy is used to address the remarkable persistence of the native Arrhenius dependence of the 2-aminopropanol substrate radical rearrangement reaction in B12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium from physiological to cryogenic (220 K) temperatures. Two-component TEMPOL spin probe mobility in the presence of 10 mM (0.08% v/v) 2-aminopropanol over 200-265 K demonstrates characteristic concentric aqueous-cosolvent mesodomain and protein-associated domain (PAD, hydration layer) solvent phases around EAL in the frozen solution. The mesodomain formed by the relatively small amount of 2-aminopropanol is highly confined, as shown by an elevated temperature for the order-disorder transition (ODT) in the PAD (230-235 K) and large activation energy for TEMPOL rotation. Addition of 2% v/v dimethylsulfoxide expands the mesodomain, partially relieves PAD confinement, and leads to an ODT at 205-210 K. The ODT is also manifested as a deviation of the temperature-dependence of the EPR amplitude of cob(II)alamin and the substrate radical, bound in the enzyme active site, from Curie law behavior. This is attributed to an increase in sample dielectric permittivity above the ODT at the microwave frequency of 9.5 GHz. The relatively high frequency dielectric response indicates an origin in coupled protein surface group-water fluctuations of the Johari-Goldstein β type that span spatial scales of ∼0.1-10 Å on temporal scales of 10-10-10-7 s. The orthogonal EPR spin probe rotational mobility and solvent dielectric measurements characterize features of EAL protein-solvent dynamical coupling and reveal that excess substrate acts as a fluidizing cryosolvent to enable native enzyme reactivity at cryogenic temperatures.

Development of food grade O/W nanoemulsions as carriers of vitamin D for the fortification of emulsion based food matrices: A structural and activity study

Oil-in-water (O/W) edible nanoemulsions were developed and studied as carriers of vitamin D (D3: cholecalciferol) to be used for the fortification of dairy emulsions. The nanoemulsions were produced using polysorbate 20, soybean lecithin and their mixtures as emulsifiers. The dispersed oil phase consisted of either soybean oil or mixtures of the oil with cocoa butter. Coarse emulsion premixes were passed through high pressure homogenizer (800–1000 bar) to produce fine droplets. The influence of system composition and preparation conditions on the stability of the nanoemulsions was investigated using Dynamic Light Scattering (DLS). Nanoemulsions containing oil droplets with mean diameters <200 nm and narrow particle size distributions (PdI < 0.3) were obtained. Different concentrations of vitamin D3 ranging from 0.1 to 0.5 μg/mL were encapsulated in the oil cores of selected stable nanoemulsions and their physical stability was evaluated by the maintenance of size distribution for several weeks. Interfacial properties of empty and vitamin enriched nanoemulsions were studied using Electron Paramagnetic Resonance (EPR) spectroscopy and an amphiphilic spin probe. Spin probe's mobility was decreased upon increase of systems complexity and also the presence of vitamin D3. Moreover, partial vitamin's embedment in the interface thus rendering it more fluid was evidenced in systems stabilized by polysorbates. Finally, whole-fat milk was fortified with the aforementioned vitamin enriched nanoemulsions and remained stable to particle growth and gravitational separation for at least ten days. Antiradical properties of encapsulated cholecalciferol were investigated using an EPR approach.

On the mutual relationships between spin probe mobility, free volume and relaxation dynamics in organic glass-formers: Glycerol

The rotation dynamics of the spin probe TEMPO in glycerol from ESR is compared with the ortho-positronium (o-Ps) annihilation from PALS and interpreted using the relaxation dynamics from BDS. Rotation time scale within the slow motion regime exhibits two Arrhenius regions with the characteristic ESR temperature, TX1τ, close to the characteristic PALS temperature, Tb1L, which is related to the secondary β process above Tg. Next, a slow to fast motion regime transition at the characteristic ESR temperature, Tcτ, close to the characteristic PALS temperature, Tb2L, followed by non-Arrhenius fast motion regime region is fully coupled with the primary α process.

Влияние пропиленгликоля и полиэтиленгликоля с молекулярной массой 1500 на микровязкость мембран эритроцитов

Electron paramagnetic resonance of spin probes was used to study the effect of low and high molecular weight CPAs on microviscosity of erythrocyte membranes. Research data on rotational mobility of the fatty acid spin probes in erythrocyte membranes after 5 min incubation in 15 % propylene glycol solution indicate the absence of strong changes in membrane microviscosity. Introduced polyethylene glycol with m. w. 1500 (PEG-1500) at a final concentration of 15% has been shown to increase the membrane microviscosity at 60 min exposure. This is explained by the fact that the molecules of PEG-1500 do not pass inside the cells and are localized on the membrane surface to cause immobilization and inhibiting the rotational mobility of the spin probe. In the initial period of time under these concentrations of PEG-1500 the injury of liquid crystal membrane structure and disorder of the protein-lipid interactions in the membrane prevail over the dehydration of cells. Probl Cryobiol Cryomed 2016; 26(1):35-44.

Formulation and characterization of food-grade microemulsions as carriers of natural phenolic antioxidants

Food-grade W/O microemulsions based on lecithin, caprylic/capric triglycerides, isopropyl myristate, alcohols and water were formulated and structurally characterized to be used as potential carriers of natural food antioxidants. Different well-known food antioxidants including gallic acid, p-hydroxybenzoic acid, protocatechuic acid and tyrosol were successfully encapsulated in the aqueous cores of the microemulsions. A pseudo-ternary phase diagram was constructed to determine the extent of the monophasic area that corresponds to an inverted type microemulsion. Apparent hydrodynamic diameter measurements of empty and loaded microemulsions were performed using dynamic light scattering (DLS) and swollen micelles with diameters smaller than 10 nm were detected. Interfacial properties of the microemulsions were studied by electron paramagnetic resonance (EPR) spectroscopy employing the nitroxide spin probe 5-doxylstearic acid (5-DSA). A small increase in spin probe mobility upon addition of the antioxidants was observed; whereas the rigidity of the surfactants was not affected. Cryogenic transmission electron microscopy (Cryo-TEM) indicated the existence of entangled thread-like reversed micelles. Finally, the investigated phenolics were assessed and compared for their radical scavenging activity using an EPR approach based on free radicals. The encapsulated gallic acid showed the highest antioxidant activity (0.93 mM trolox equivalents) as compared to other antioxidants assessed within the frame of this study.

The Myosin Inhibitor Blebbistatin Stabilizes the Super-Relaxed State in Skeletal Muscle

The super-relaxed state of myosin (SRX), in which the myosin ATPase activity is strongly inhibited, has been observed in a variety of muscle types. It has been proposed that myosin heads in this state are inhibited by binding to the core of the thick filament in a structure known as the interacting-heads motif. The myosin inhibitor blebbistatin has been shown in structural studies to stabilize the binding of myosin heads to the thick filament, and here we have utilized measurements of single ATP turnovers to show that blebbistatin also stabilizes the SRX in both fast and slow skeletal muscle, providing further support for the proposal that myosin heads in the SRX are also in the interacting-heads motif. We find that the SRX is stabilized using blebbistatin even in conditions that normally destabilize it, e.g., rigor ADP. Using blebbistatin we show that spin-labeled nucleotides bound to myosin have an oriented spectrum in the SRX in both slow and fast skeletal muscle. This is to our knowledge the first observation of oriented spin probes on the myosin motor domain in relaxed skeletal muscle fibers. The spectra for skeletal muscle with blebbistatin are similar to those observed in relaxed tarantula fibers in the absence of blebbistatin, demonstrating that the structure of the SRX is similar in different muscle types and in the presence and absence of blebbistatin. The mobility of spin probes attached to nucleotides bound to myosin shows that the conformation of the nucleotide site is closed in the SRX.

Вплив зберігання тканини плаценти при –20°С на властивості її водно-сольових екстрактів

Research was aimed to study the effect of human placenta tissue storage at –20°C on the features of procured from it aqueous-saline extracts and their certain fractions. The methods of gel-chromatography, optical spectroscopy and spin probe EPR were used in the research. Spin probe EPR demonstrated the exposure of erythrocytes with fraction of human placenta extracts below 5 kDa as not affecting the microviscosity of erythrocyte cytosol. Along with that we observed the modification of temperature dependence of spin probe mobility under exposure with the same fraction of placenta extracts, stored for 6 months at –20°C. This correlated with a decrease in acid resistance of erythrocytes under the effect of this fraction. Probl Cryobiol Cryomed 2014; 24(1):28–37.

Orientational order of guest molecules in aligned liquid crystal as measured by EPR and UV–vis techniques

Orientational order of guest molecules in aligned liquid crystal 4-cyano-4′-pentylbiphenyl (5CB) is studied via optical dichroism and electron paramagnetic resonance (EPR) spectra measurements. The guest molecules used are bifunctional molecules bearing paramagnetic nitroxide group and photochromic azobenzene moiety. The bifunctional probe with rigidly bonded nitroxide and azobenzene moieties was found to align as a whole, while flexible long spacer between the moieties provides independent alignment for the nitroxide and azobenzene parts. Intermolecular interactions responsible for the alignment of azobenzene and nitroxide moieties of the probe molecules are discussed. The molecules with cis-configuration of azobenzene moiety are able to align in the liquid-crystalline medium, but to a lesser extent than the molecules with trans-configuration. Directions of orientational axes and characteristics of rotational mobility of spin probes are determined. Second, fourth and, in some cases, sixth rank order parameter values are found.

Local mobility of micelles of new cationic surfactants and their interactions with hydrophobically modified polyacrylamides

The local dynamics and organization of micelles of new long-chain cationic surfactants with saturated hydrocarbon fragments (from C16 to C22) are investigated via the EPR spin-probe technique. The local mobility of spin probes in the hydrocarbon core of a micelle changes insignificantly, while the order parameter noticeably increases with lengthening of the hydrocarbon fragment of the surfactant molecule. The specific features of the interaction of the surfactants with network junctions of the gels formed by two types of hydrophobically modified polyacrylamides—either containing charged groups (sodium acrylate) in the backbone or lacking these groups—are studied. In both cases, the local mobility of network junctions of the gel increases after the introduction of the surfactant (C18). Moreover, for surfactant with a long alkyl group (C2), the microscopic viscosity of the gel based on the uncharged polymer decreases, although the local mobility of the network junctions increases. Possible causes of the observed specific features are discussed.

Fluorescence and ESR spectroscopy studies on the interaction of isoflavone genistein with biological and model membranes

Genistein (5,7,4′-trihydroxyisoflavone) the common soy beans isoflavone has attracted scientific interest due to its antioxidant, estrogenic, antiangiogenic and aniticancer activities. The aim of the present study was to investigate the interaction of genistein with biological (erythrocyte) and model membranes (dimyristoyl- and dipalmitoylphosphatidylcholine). Using Laurdan and Prodan as fluorescent probes, we demonstrated phase behavior and membrane fluidity changes induced by genistein. ESR spectroscopy revealed alterations caused by genistein in membrane domains structure and mobility of spin probes with free radicals located at different depths of membrane. The method of ESR spectra decomposition and computer simulation of the recorded spectra were used in order to visualize domain coexistence by GHOST condensation method. Fluorescence and ESR spectroscopy experiments performed at different temperatures enabled us to observe the effect of isoflavone on phospholipid bilayers in either gel or liquid crystalline phase. It was concluded that genistein preferentially intercalated into lipid headgroup region, to some extent into polar–apolar interface and only in minimal degree into hydrophobic core of the membrane. According to our best knowledge this is the first study on modification of domain structure of membranes by genistein.

Microemulsions based on virgin olive oil: A model biomimetic system for studying native oxidative enzymatic activities

Water-in-oil (w/o) microemulsions, composed of virgin olive oil as the continuous oil phase, a mixture of lecithin and propanol as emulsifiers and water as the dispersed phase, have been successfully used as model biomimetic media to carry out enzymatic reactions that may naturally occur in olive oil. To evaluate the size of the dispersed polar domains as a function of water content, dynamic light scattering (DLS) measurements were carried out and radii in the range of 60–130 nm were observed. The polarity of the interior of these polar domains was studied by electron paramagnetic resonance (EPR) spectroscopy using a polar spin probe. Up to certain water content (1% w/w) both polarity and spin probe's mobility were affected by water content of the system and then remained unaltered. Oxidative enzymatic reactions using a native olive peroxidase (POD) also present in virgin olive oil, and horseradish peroxidase (HRP) were carried out in w/o microemulsions using 2,2′-azino-bis[3-ethylbenzo-thiazoline-6-sulfonic acid] (ABTS) and gallic acid as substrates. Enzymatic activities were affected by the restricted environment of the nanoreactors and also the different polarity of the medium leading to comparable initial velocity values for both substrates.

Rotational and Translational Mobility of Nitroxide Spin Probes in Ionic Liquids and Molecular Solvents

Rotational and translational movements of 1-oxyl-2,2,6,6-tetramethyl-4-oxypiperidine (TEMPOL) spin probe in the room temperature ionic liquid (RTIL) 1-octyl-3-methylimidazolium tetrafluoroborate (omimBF4) and in two molecular solvents, 1-propanol and isopropyl benzene (cumene), have been studied by X-band electron paramagnetic resonance (EPR) spectroscopy. Rotational correlation times τ c of spin probes and the intermolecular spin exchange rate constants k e were measured from EPR spectra at different temperatures and TEMPOL concentrations, and compared with the published data. The τ c values were calculated both by known equations and from the EPR spectra simulation. Rotation movements of TEMPOL in omimBF4 cannot be described by the model of the isotropic Brownian diffusion, which is valid for conventional solvents. The correct modeling of EPR spectra in RTIL can be achieved with the assumption of different rotational mobility of the spin probe around different molecular axes. The rotational, D rot, and translational, D tr, diffusion coefficients were calculated from τ c and k e values. The Debye–Stokes–Einstein law is valid in all three solvents while the dependence of D tr on T/η is not linear in Stokes–Einstein coordinates. The effective activation energy E rot a of the rotational movements in omimBF4 is noticeably higher than the corresponding values for conventional solvents, while the effective activation energies E tr a of the translational movements are comparable in all solvents studied.

Mobility of spin probes in viscous cyclodextrin solutions

TEMPO-based spin probes and beta-cyclodextrin form 1 ratio 2 guest-host complexes in viscous aqueous glycerol solutions at low temperatures. The spin probe mobility in these complexes is much higher than in the bulk glassy solution. The similar tumbling rates of the spin probe in viscous host-guest complexes and in solvent-free complexes suggest that the complexation effectively shields the nitroxide unit from the solvent molecules. This is consistent with the low activation barrier for molecular tumbling of immobilised label.

Spin probe mobility in relation to free volume and relaxation dynamics of glass‐formers: A series of spin probes in poly(isobutylene)

AbstractWe present the dynamics of a series of three paramagnetic molecules of different volume, mass, and shape in amorphous glass‐forming polymer poly(isobutylene) (PIB) as investigated by means of electron spin resonance (ESR) technique. The reorientation behavior of spin probes is related to the ortho‐positronium (o‐Ps) annihilation in PIB from positron annihilation lifetime spectroscopy (PALS) and the extracted free volume information. It is also related to the dynamic data of PIB from broadband dielectric spectroscopy (BDS), neutron scattering (NS), and nuclear magnetic resonance (NMR) spectroscopy from literature. In the case of the smallest spin probe, 2,2,6,6‐tetramethyl‐1‐piperidinyloxy (TEMPO), a discontinuous course of the spectral parameter 2Azz′ versus T dependence was observed and the subsequent phenomenological model‐free analyses of the spectral parameter, 2Azz′ versus T, as well as of the correlation time, τc, versus 1/T plots provided the characteristic ESR temperatures ($T_{X1}^{2A_{zz}}$, T50G, $T_{X2}^{2A_{zz}}$) and (T, T, T). These characteristic ESR temperatures were found to be consistent with the characteristic PALS temperatures: T, T = T from temperature dependences of the mean o‐Ps lifetime, τ3, or the width of o‐Ps lifetime distribution, σ3, respectively. In addition, the relationships between the spin probe size, V, and the free volume hole size distributions gn(Vh) at the characteristic ESR temperatures indicate the significant influence of the free volume fluctuation at the crossover from slow to rapid regime as well as within the rapid motional regime. On the other hand, the two larger spin probes exhibit a rather continuous 2Azz′–T plots with the respective T50G's lying in the vicinity of T independently of their volume, mass and shape, suggesting the common origin of underlying process controlling this T50G transition. Finally, these mutual PALS and ESR findings were compared with the known dynamic behavior of PIB which suggest that the dynamics of the TEMPO and the larger spin probes are related to free volume fluctuation associated with primary α ‐ and secondary β processes, respectively. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 47: 1058–1068, 2009

Heterogeneity of thylakoid membranes studied by EPR spin probe

A lipophilic nitroxyl radical, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 1-adamantylacetate, has been applied to EPR spin probe study of chloroplasts and subchloroplast fragments of different types. The latter originate from grana and the grana core regions. The binding of the spin probe to the membranes was revealed by specific changes in a shape of the EPR spectra. A share of membrane-bound spin probe was different for chloroplasts and subchloroplast fragments, as well as its rotational correlation time and apparent enthalpy and entropy activation of nitroxide rotational motion. The binding of the spin probe induced a significant decrease in the amount of the oxidized P700 and changes in the kinetics of its light oxidation and dark recovery. This suggests that one of the sites of nitroxyl radical binding is the nearest surrounding of the pigment-protein complexes of Photosystem I (PSI). Distinctions in mobility of spin probe immobilized by chloroplasts and their fragments can be caused by the different environment of the PSI complexes located in various regions of thylakoid membranes.

Influence of silybin on biophysical properties of phospholipid bilayers

Silybin (silibinin) is major biologically active flavonolignan extracted from milk thistle (Sylibum marianum). Its biological activities include hepato-protection, anticancer properties, and antioxidant- and membrane-stabilizing functions. Although membranes are postulated to be one of the cellular targets for silybin, little is known about its interaction with phospholipid bilayers. In the present work, the interactions of silybin with phosphatidylcholine bilayers were studied in detail using fluorescence spectroscopy, microcalorimetry and electron spin resonance techniques. The results showed that silybin interacted with the surface of lipid bilayers. It affected the generalized polarization of the fluorescent probe Prodan, while not influencing the more deeply located Laurdan. Silybin lowered the main phospholipid phase transition temperature as judged by microcalorimetry, and caused the immobilization of spin probe Tempo-palmitate located on the surface of membranes. The mobility of spin probes 5- and 16-doxyl stearic acid was not affected by silybin. Silybin-induced quenching of 1,6-diphenyl-1,3,5-hexatriene fluorescence indicated that some flavonoid molecules partitioned into the hydrophobic region of membranes, which did not change significantly the biophysical properties of the deeper membrane regions. Such a behavior of silybin in membranes is in accordance with its postulated biological functions and neglectable side effects of therapies using silybin.

Mobility of spin probes in ionic liquids

AbstractThe spin probes TEMPO, TEMPOL, and CAT‐1 were used to investigate microviscosity and micropolarity of imidazolium based ionic liquids bearing either tetrafluoroborate or hexafluorophosphate as anions and a variation of the substitution at the imidazolium ion. The average rotational correlation times (τ) obtained by complete simulation of the X‐band ESR spectra of TEMPO, TEMPOL, and CAT‐1 increase with increasing viscosity of the ionic liquid although no Stokes Einstein behavior is observed. This is caused by microviscosity effects of the ionic liquids shown by application of the Gierer–Wirtz theory. Interestingly, the jump of the probe molecule into the free volume of the ionic liquids is a nonactivated process. The hyperfine coupling constants (Aiso (14N)) of TEMPO and TEMPOL dissolved in the ionic liquids do not depend on the structure of the ionic liquids. The Aiso (14N) values show a micropolarity of the ionic liquids that is comparable with methylenchloride in case of TEMPO and with dimethylsulfoxide in case of TEMPOL. Micropolarity monitored by CAT‐1 strongly depends on structural variation of the ionic liquid. CAT‐1 dissolved in imidazolium salts substituted with shorter alkyl chains at the nitrogen atom exhibits a micropolarity comparable with dimethylsulfoxide. A significant lower micropolarity is found for imidazolium salts bearing a longer alkyl substituent at the nitrogen atom or a methyl substituent at C‐2. Copyright © 2006 John Wiley &amp; Sons, Ltd.

Micellar aggregation of alkyltrimethylammonium bromide surfactants studied by electron paramagnetic resonance of an anionic nitroxide

The self-aggregation process of five alkyltrimethylammonium bromide surfactants [CH3(CH2)n−1N(CH3)3Br (n = 6,8,10,12,16), CnTAB] in aqueous solution at pH = 7 has been studied by electron paramagnetic resonance (EPR) spectroscopy by employing 3-carboxy-PROXYL in its deprotonated form [2,2,5,5-tetramethyl-3-carboxypyrrolidinyloxy sodium salt, CP−] as a spin probe. In all the considered systems, the nitrogen isotropic hyperfine coupling constant of CP−, 〈AN〉, decreases and the correlation time, τC, increases with increasing surfactant molality. Concerning the surfactants with long hydrophobic tails (n = 8,10,12,16), both 〈AN〉 and τC present a slope change corresponding to the critical micellar composition, c.m.c. In these cases the τC increase can be interpreted in terms of a reduction of the spin probe mobility determined by the strong electrostatic interaction between the CP− charge and the cationic micelles' surface. Particularly, the τC values show that the microviscosity experienced by CP− in C16TAB micelles is much higher in respect to that found in the other micellar systems, thus suggesting a different structural organization of the aggregates’ surface. The 〈AN〉 decrease can be ascribed to a partial embedding of the NO moiety of CP− in the outer part of the micellar hydrophobic core. The CP− affinity for the micellar pseudo-phase has been estimated by evaluating the distribution coefficient, Kd, of the spin probe between the micelles and the aqueous medium. The Kd value increases with the length of the surfactant hydrophobic chain. Because of the short hydrophobic tail, the C6TAB aqueous mixtures exhibit a peculiar behaviour: 〈AN〉 smoothly decreases and τC of CP− increases with increasing surfactant molality without any abrupt slope change. This experimental evidence suggests that C6TAB association can be described in terms of a gradual change, with increasing surfactant molality, from solvent-mediate to direct surfactant–surfactant interactions.