Mitogens-DPPC-vesicles interaction: A spin label ESR study

Summary The electron spin resonance spectroscopy with the spin labelling technique has been used to investigate the interaction of sonicated di-palmitoyl-phosphatidylcholine (DPPC) small unilamellar vesicles with concanavalin A (Con A), phytohemagglutinin (PHA) and staphylococcal enterotoxin B (SEB) in the temperature range 0–50°C. Conventional ESR measurements show that the addition of mitogens to the vesicles suspensions up to the number of mitogens/number of vesicles ratio of 60 increases the orientational degree of order of the lipid molecules in the gel phase and the gel→liquid-crystalline phase transition temperature. No effects on the liquid-crystalline phase have been observed, also no matter for the mitogen used. The effectiveness results in the order: ConA>SEB>PHA. Saturation transfer-ESR measurements evidence more restricted rotation of the DPPC molecules in the gel phase in the presence of Con A and SEB than in the presence of PHA if compared to pure DPPC-vesicles. The effective rotational correlation time, σ_c, of the spin label 5-SASL results about one order of magnitude higher in the presence of 60 mitogen molecules/vesicle. The ESR findings suggest a mitogen-induced increase in the packing density of lipid bilayers which is attributed to a morphological change from sonicated vesicles, with a small radius of curvature, to a more planar mesophase. Con A is more effective than SEB and PHA in inducing this process. The authors of this paper have agreed to not receive the proofs for correction.     

Relaxation-based distance measurements between a nitroxide and a lanthanide spin label

Distance measurements by electron paramagnetic resonance techniques between labels attached to biomacromolecules provide structural information on systems that cannot be crystallized or are too large to be characterized by NMR methods. However, existing techniques are limited in their distance range and sensitivity. It is anticipated by theoretical considerations that these limits could be extended by measuring the enhancement of longitudinal relaxation of a nitroxide label due to a lanthanide complex label at cryogenic temperatures. The relaxivity of the dysprosium complex with the macrocyclic ligand DOTA can be determined without direct measurements of longitudinal relaxation rates of the lanthanide and without recourse to model compounds with well defined distance by analyzing the dependence of relaxation enhancement on either temperature or concentration in homogeneous glassy frozen solutions. Relaxivities determined by the two calibration techniques are in satisfying agreement with each other. Error sources for both techniques are examined. A distance of about 2.7 nm is measured in a model compound of the type nitroxide–spacer–lanthanide complex and is found in good agreement with the distance in a modeled structure. Theoretical considerations suggest that an increase of the upper distance limit requires measurements at lower fields and temperatures.     

Procain interaction with DPPC multilayers: an ESR spin label investigation

A spin label electron spin resonance (ESR) study has been carried out on the interaction between the local anesthetic procaine (PRC) and multilamellar dispersions of di-palmitoylphosphatidylcholine (DPPC). The investigation has been performed either in the gel or in the liquid crystal phase of DPPC varying the anesthetic/DPPC molar ratio as well as the pH of the dispersion medium and using spin labeled DPPC molecules (5- and 16-PCSL) and the di-tert-butyl nitroxide (DTBN) spin probe. On increasing the anesthetic concentration in the dispersion medium up to PRC/DPPC molar ratio of 33 mol.%, a lyotropic transition of the bilayer from the lamellar gel phase L_β, to the interdigitated L_βi one is observed either at pH 5.0 or at pH 10.0. At intermediate pH values, about pH 7.0, such a PRC concentration dependent transition is absent. A decrease of the partition of the DTBN molecules between the dispersion medium and the fluid hydrophobic phase of DPPC multilayers is observed at pH 5.0 with anesthetic concentration, while an opposite behaviour is evidenced at pH 10.0. The pre- and main-phase transition temperatures of the DPPC multilayers are also affected although to different extent. These results are interpreted in terms of adsorption of the PRC molecules at the surface of the model membrane at the extreme pH values investigated, while at about neutral pH values they intercalate among the DPPC molecules. The protonation state of procaine plays an important role as it determines the way of surface adsorption of the PRC molecules in the neutral form at the high pH value and in the charged cationic form at pH 5.0.     

Some EPR spin probe and spin label studies of polymer systems

The application of EPR spin probe and spin label technique for solving the two problems of polymer physical chemistry is considered. The first problem is the determination of conformational state and chain sizes in amorphous solid polymers. This determination is based on the analysis of the intramolecular dipolar broadening of EPR spectra of spin labelled macromolecules in glassy solvents or in the bulk of unlabeled polymers at 77 K. The second problem is the determination of molecular mobility and structure of two polymer colloid systems: (a) the complex of colloidal silica and synthetic polycation macromolecule and (b) polymer-surfactant micellar organized systme.     

Analysis of nitroxide spin label motion in a protein-protein complex using multiple frequency EPR spectroscopy

X- and W-band EPR spectra, at room and low temperatures, are reported for nitroxide spin labels attached to cysteine residues selectively introduced into two proteins, the DNase domain of colicin-E9 and its immunity protein, Im9. The dynamics of each site of attachment on the individual proteins and in the tight DNase-Im9 complex have been analysed by computer simulations of the spectra using a model of Brownian dynamics trajectories for the spin label and protein. Ordering potentials have been introduced to describe mobility of labels restricted by the protein domain. Label mobility varies with position from completely immobilised, to motionally restricted and to freely rotating. Bi-modal dynamics of the spin label have been observed for several sites. We show that W-band spectra are particularly useful for detection of anisotropy of spin label motion. On complex formation significant changes are observed in the dynamics of labels at the binding interface region. This work reveals multi-frequency EPR as a sensitive and valuable tool for detecting conformational changes in protein structure and dynamics especially in protein-protein complexes.     

Submillimeter-wave ESR measurements of low-dimensional quantum spin systems

Temperature-dependent electron spin resonance (ESR) measurements of two low-dimensional quantum spin systems, theS = 1/2 spin-ladder system Cu₂(1,4-diazacycloheptane)₂Cl₄ (Cu₂(C₅H₁₂N₂)₂Cl₄) and the gaplessS = 1 bond-alternating one-dimensional antiferromagnettrans-Ni(333-tet)(N₃)(ClO₄), have been performed. X-band electron spin resonance (ESR) measurements of single-crystal Cu₂(C₅H₁₂N₂)₂Cl₄ show the increase of linewidth and anisotropicg-shifts below 8 K similar to those known for one-dimensional antiferromagnet. On the other hand, Ni(333-tet)(N₃)(ClO₄) has broad line-width and ESR has been observed for the first time by our high-field ESR. Its linewidth increases as the temperature is decreased, while theg-shift seems to be isotropic and theg-value decreases as the temperature is decreased. Thisg-shift can be connected to the quantum fluctuation of the system.     

Spin concentration in a possible ESR dosimeter: An electron spin echo study on X-irradiated ammonium tartrate

Several single crystals and powder samples of ammonium tartrate, recently proposed as a possible ESR dosimeter, have been X-irradiated with different doses. The total radical concentration has been determined by quantitative cw ESR, by comparison with a standard. The samples have been studied by electron spin echo spectroscopy. The two-pulse echo decay has been obtained and simulated by a single exponential function for different values of the microwave power of the pulses and for different pulse lengths. The dependence of the phase memory time TM on the microwave power has been exploited to get information on the contribution of the instantaneous diffusion to spin dephasing. At room temperature in the range of radical concentrations of 10(18)-10(19) spins/cm3 the instantaneous diffusion is the dominant spin dephasing mechanism. The linear dependence of the instantaneous diffusion on the total concentration of the radicals is in agreement with the theory. From the latter result we conclude that the average radical-radical distance corresponds to a random distribution of the radicals in the matrix. A simple method of measuring the radical concentration by the ESE decays in powder samples of irradiated ammonium tartrate is described.     

Nuclear magnetic resonance and spin relaxation in biological systems

Proton nuclear spin-lattice relaxation in biological systems is generally distinguished from that in inorganic systems such as rocks by the presence of locally disordered macromolecular environments. Rapid exchange of readily observed labile small molecules among differently oriented macromolecular sites generally nearly averages the spectral anisotropies in the small molecule resonances. The biological tissue is generally distinguished from the inorganic matrix by the presence of a significant population of protons in the solid components that are well connected by dipolar spin couplings. Magnetic coupling between the solid and the liquid components generally dominates the magnetic field dependence of the spin-lattice relaxation rates observed in the small molecule components which is generally described by a power law in the Larmor frequency. Recent theory involving a modification of the spin-phonon class of relaxation mechanism provides a quantitative understanding of these data in terms of the dynamics of the chain molecules generally present in the solid spin systems, folded proteins for example.     

A spatiotemporal study on the distribution of intraperitoneally injected nitroxide radical in the rat head using an in vivo ESR imaging system

We have developed a rapid-scan in vivo electron spin resonance (ESR) imaging system operating at 700 MHz based on an air-cored two-coil Helmholtz designed resistive magnet. Using this system, we performed ESR-CT for the intraperitoneally injected nitroxide radical, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl, in the rat head. The imaging data were collected over the time course range from 5 to 47 min after injection at an interval of 3 min and a series of ESR-CT images were reconstructed at the same slice plane (1 cm anterior to interaural line). The series of ESR-CT images thus obtained by rapid scans provided detailed spatiotemporal information on the distribution of the injected nitroxide radical in the rat head. The brain was imaged as a nitroxide-deficient area while the blood vessels and/or extracranium tissues as a nitroxide-rich area. During periods when high intensities of ESR signals were maintained, spots of nitroxide-accumulation were imaged at the central part of the brain. The spots were assigned to the middle sized blood vessels in the brain.     

A novel approach to the simulation of nitroxide spin label EPR spectra from a single truncated dynamical trajectory

A simple effective method for calculation of EPR spectra from a single truncated dynamical trajectory of spin probe orientations is reported. It is shown that an accurate simulation can be achieved from the small initial fraction of a dynamical trajectory until the point when the autocorrelation function of re-orientational motion of spin label has relaxed. This substantially reduces the amount of time for spectra simulation compared to previous approaches, which require multiple full length trajectories (normally of several microseconds) to achieve the desired resolution of EPR spectra. Our method is applicable to trajectories generated from both Brownian dynamics and molecular dynamics (MD) calculations. Simulations of EPR spectra from Brownian dynamical trajectories under a variety of motional conditions including bi-modal dynamics with different hopping rates between the modes are compared to those performed by conventional method. Since the relatively short timescales of spin label motions are realistically accessible by modern MD computational methods, our approach, for the first time, opens the prospect of the simulation of EPR spectra entirely from MD trajectories of real proteins structures.     

Partition coefficient of small spin probes: An improved ESR determination method

We report on the improved determination of the partition coefficient,P _e, of the small hydrophobic spin probe di-tert-butyl nitroxide (DTBN) between the aqueous and the fluid hydrophobic phase of dipalmitoylphosphatidyl choline (DPPC) multilayers, made in terms of the second integral evaluation of the isotropic ESR resonance lines. The calculation of the second integral was carried out taking into account both the width and amplitude of the resonance lines and a shape factor related to the degree of inhomogeneous line broadening. The expression ofP _c obtained in this way has been tested by computer simulation using Voightian lineshapes for the ESR absorption resonance lines. The method has been applied to the determination of the degree of cooperativity of the gel → liquid-crystal phase transition of DPPC multilamellae, and the results were compared with those obtained using the traditional expression ofP _e. Finally, the calculation of the cooperativity parameter of the main phase transition of DPPC multilayers results improved of about 70% by using the new expression ofP _c.     

Influence of general-inhalation anesthetics on DPPC vesicles: a spin label study

Summary Electron spin resonance spectroscopy has been used to investigate the effects of three inhalation anesthetics,i.e. halothanechloroform-diethyl ether, on the thermomotropic behaviour of the polar interface and hydrophobic region as well as of DPPC-sonicated unilamellar vesicles. The study performed in the temperature range (25÷45)°C and for DPPC-to-anesthetic molar-concentration ratios of 4∶1, 2∶1 and 1∶1 shows that anesthetics, for their mechanisms of action, can be grouped in two classes: halothane and chloroform belong to one class, while diethyl ether to the other one. The former two downshift the main phase transition temperature,T _t, of DPPC in a remarkable way, whilst the latter one influences the gel→liquid crystalline-transition temperature in a less extent. TheT _t downshift depends on anesthetic concentration and the effectiveness in perturbing the bilayer order results halothane>chloroform≫ diethyl ether. Two microenvironments with a different degree of anisotropic motion are found in the hydrophobic region of vesicles in the absence of anesthetics. Halothane and chloroform affect the region more or less in a similar way which, in turn, results to be different from the action exerted on the microenvironments by diethyl ether. To speed publication, the authors of this paper have agreed to not receive the proofs for correction.     

Pair exchange interaction in a crystalline nitroxide radical ESR study of dimers in the triplet state

The dimerization of the 9-aza-bicyclo (3,3,1) nonan-3-one-9-oxyl in the solid state is investigated by use of ESR spectroscopy. The ESR spectrum of a single crystal is characteristic of symmetric pairs of exchange-coupled radicals in a thermally accessible triplet state. The presence of well-resolved hyperfine structure is evidence for strongly localized excitations with a jumping rate lower than 10⁷ Hz.

The ESR spectrum is well described by the spin hamiltonian

At 35 GHz the observed splitting of the m _s=+ 1?0 transition has been found to be slightly different from that of the m _s=0?-1 one; this anomaly is explained by the mixing of the m _s electronic states.

The parameters and the principal directions of the zero-field splitting, spectroscopic and hyperfine tensors are determined and discussed. The principal directions of the dipolar tensor indicate a nearly equal spin density on the nitrogen and oxygen atoms; from the fine structure parameters D and E, determined to be (-0·0723 ± 0·0005) cm^-1 and (-0·0044±0·0003) cm^-1 at T=293 K respectively, it is suggested that the unpaired electron is partly delocalized on the molecule.

The singlet-triplet energy gap (J) and the zero-field splitting parameters are shown to be linearly temperature-dependent. These variations with temperature are attributed to the thermal expansion of the crystal lattice.     

Electrostatic interaction on purple membrane: a spin label study onpH and ionic-strength effects

Summary The effects ofpH and ionic strength on the thermotropic phase transition behaviour of lipids of purple membrane have been investigated by electron spin resonance spectroscopy using the spinlabelling technique. The spin label 5-nitroxyl stearic acid located into the polar-head region of the lipid membrane shows atpH 5.0 and in the absence of 1:1 electrolyte an ordered → fluid phase transition at the temperature ofT _t≃40°C which decreases to ≃36°C when thepH of the dispersion is increased to 9.0. The down-shift ofT _t is more pronounced if the purple membrane is dispersed at bothpHs in 3 M NaCl. The shifts observed strongly suggest that ions affect the electrical properties of the negatively charged lipids of the membrane and, therefore, the structure of the polar region. On the other hand, the system of hydrocarbon chains of purple membrane results unaffected by charge variations on the membrane surface induced bypH and ionic-strength variations.

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Riassunto Mediante risonanza di spin elettronico e la tecnica della marcatura di spin sono stati studiati gli effettti delpH e della forza ionica sulla transizione di fase termotropica dei lipidi della membrana purpurea del batterio H. halobium. Lo spin-label 5-NSA localizzato nella regione polare dei lipidi di membrana mostra apH 5.0 ed in assenza di elettroliti 1:1 una transizione di fase stato ordinato → stato fluido alla temperaturaT _t≃40°C che scende a ≃36°C quando ilpH della dispersione è aumentato a 9.0. Lo spostamento osservato nella temperatura di transizione è piú pronunciato se la membrana purpurea è dispersa in una soluzione 3 M NaCl ai due valori dipH. L'effetto osservato indica che gli ioni influenzano le proprietà elettriche dei lipidi di membrana carichi negativamente e di conseguenza la struttura della regione polare del doppio strato. Al contrario, il nucleo idrofobico del doppio strato non risulta influenzato nè da variazioni dipH che di forza ionica.

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Резюме ВлияниеpH и ионной силы раствора на поведение липидов пурпурной мембраны при термотропных фазовых переходах исследовалось методом электронного спинового резонанса с использованием методики спиновой метки. Введение меченой 5-нитроксил стеариновой кислоты в области полярных головок липидов мембраны обнаруживает приpH=5.0 и в отсутствии 1:1 электролита ?упорядоченный → жидкость? фазовый переход при температуреT _t≃40°C, которая уменьшается до ≃36°C, когдаpH увеличивается до 9.0 Сдвиг температурыT _t наиболее резко выражен, если пурпурная мембрана находится при тех ЗеpH в 3 M растворе NaCl. Наблюдаемые сдвиги указывают, что ионы влияют на электрические свойства отрицательно заряженных липидов мембраны и, следовательно, на структуру полярной области. С другой стороны, система гидрокарбонатных цепочек пурпурной мембраны не подвержена воздействию изменений заряда на поверхности мембраны, которые обусловлены изменениямиpH и ионной силы раствора.

     

Hydration and protein dynamics: an ESR and ST-ESR spin labelling study of human serum albumin

Human serum albumin has been studied at low hydration level by the ESR spin labelling technique, under the assumption that a covalently bound spin-label is a reporter of the protein internal dynamics. At room temperature, the presence of a double component signal allowed us to monitor the influence of increasing hydration level on internal protein dynamics as well as on the superficial water dynamics. The ESR results have shown that the first 20 g of water per 100 g of protein activate the internal protein dynamics and that superficial water dynamics starts at higher hydration values. ESR experiments at low temperature have shown that at ?160°C ?T??80°C, the label experiences an increasing environmental polarity with increasing temperature in the samples with hydration values higher than about 20 g of water per 100 g of protein. The results are discussed in connection with both conformational substates of the protein and hydration water dynamics.     

Electrolyte interaction with DPPC vesicles: An ESR study

Summary The partition of the spin probe TEMPO between the fluid lipid phase of single-walled vesicles of dipalmitoylphosphatidylcholine and the aqueous bulk solution have been used to investigate the interaction of monovalent ions with polar head of neutral phospholipids. The study has been performed by electron spin resonance (ESR) spectroscopy in the temperature range of (20÷60)°C and in the presence of (0÷3) M 1∶1 electrolyte. In the absence of electrolyte the spin probe TEMPO reveals the characteristic order→disorder DPPC main phase transition atT _m≈37°C, while the pretransition occurs atT _p≈27.5°C. On increasing the ionic strength of the dispersion medium it results for the partition coefficient,P _C, that, at each temperature,P _C(3)>P _C(2)>P _C(1)>P _C(0). Correspondingly, the pretransition disappears and theT _m value downshifts from ≈37°C with 0 M electrolyte to ≈34°C with 3M salt in the order:T _m(3)>T _m(2)>T _m(1)>T _m(0). The results suggest an increase in the net surface charge density of vesicles due to high ionic-strength values. The alteration of the electric interactions occurring into the polar zone of DPPC bilayer reduces the hindrances which, in turn, favour the enhancement of TEMPO partitioning in the hydrophobic core of phospholipid bilayers. The authors of this paper have agreed to not receive the proofs for correction.     

Kinetics of the polarization transfer in spatially disordered spin systems: Depolarization of impurity beta-active nuclei

We performed theoretical and experimental studies of the delocalization of the nuclear polarization of impurity beta-active nuclei ⁸Li in the spatially disordered system of ⁶Li nuclei in LiF single crystals. The process is controlled by the dipole-dipole interaction of nuclear magnetic moments with other nuclei (⁷Li, ¹⁹F) in the crystal. It is effective in a wide range of magnetic fields H ₀ = 150–3000 G as a result of a unique proximity of the g-factors of ⁸Li and ⁶Li nuclei: (g(⁸Li) − g(⁶Li))/g(⁶Li) = 0.0057. The kinetics of the ⁸Li depolarization is measured for an ⁶Li concentration of 0.15%–10.06% and in the field H ₀ of 200, 692 and 1210 G. A satisfactory explanation of the results is obtained on the basis of a numerical-analytical simulation of the process.     

Rates of rotational diffusion and Heisenberg spin exchange as obtained from CW ESR and ESR tomographic experiments on model systems and human skin

The purpose of the present paper is to describe possibilities and limitations for the determination of the rates of rotational diffusion and Heisenberg spin exchange, obtained from continuous-wave electron spin resonance (cw ESR) and ESR tomographic experiments. Model systems including nitroxides as paramagnetic reporter molecules have been examined in order to verify data, which have been obtained from cw ESR and ESR tomography. This has been done with particular emphasis on checking the influence of concentration, temperature, and viscosity on the spectral-spatial properties. These findings have been applied to the evaluation of penetration and permeation studies on human skin. The extracted full spectral information from ESR tomography allows the determination of the above mentioned dynamic parameters for model systems of definite geometry and for samples of human skin. It has been found that the signal-to-noise ratio is critical for all discussed applications.     

Investigation of biological systems by high resolution 2 mm wave band ESR

The application of the high resolution ESR technique for investigation of various biological systems is shown. The advantages of the technique in the study of structural, conformational and dynamic characteristics are exemplified by spin labeled human serum albumin, egg lysozyme, liposome membranes, inverted micelles, α-chymotrypsin, cotton fiber and cellulose. The polarity of microenvironment and the mechanisms of molecular mobility of the objects under study are determined. The combination of high resolution and saturation transfer techniques is shown to give the detailed analysis of very slow molecular motions in biological objects. Peroxide radicals in biosystems are identified by the ESR spectra of the 2 mm wave band.     

Electron spin resonance (ESR) dating and ESR applications in quaternary science and archaeometry

The scope of application of ESR spectroscopy has greatly expanded with the advent of its widespread use in radiation exposure dating around 1980 and its use in retrospective dosimetry since the Chernobyl disaster in 1986. Few fields of study are of such breadth that they span topics as diverse as dating of human origins, volcanic activity, cave deposits and earthquakes, while also providing prognoses for radiation accident victims. Between 1945 and 1975 ESR was mainly used to define the nature of paramagnetic defects in crystalline and amorphous materials, which laid the foundation for its use in applied areas in Quaternary geology, archaeometry and accident dosimetry. This review chronicles the development of the use of ESR in applied science since 1975, with particular emphasis on the state of the art in the period 1987–1997. The first part of the review focuses mainly on the range of applications for datable materials: tooth enamel (Section 2), calcite (Section 3) and quartz (Sections 4–9), while the second part comprises the areas of retrospective dosimetry (Section 10) and new applications (Section 11). The review concludes (Appendix A) with an introduction to the physical basis and assumptions involved in ESR dating, and compilations of valuable reference works for students and workers in this field.