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At least 73 records · Page 4Linked to original sources
PubMed1995

Electron-electron spin-spin interaction in spin-labeled low-spin methemoglobin.

Nitroxyl free radical electron spin relaxation times for spin-labeled low-spin methemoglobins were measured between 6 and 120 K by two-pulse electron spin echo spectroscopy and by saturation recovery electron paramagnetic resonance (EPR). Spin-lattice relaxation times for cyano-methemoglobin and imidazole-methemoglobin were measured between 8 and 25 K by saturation recovery and between 4.2 and 20 K by electron spin echo. At low temperature the iron electron spin relaxation rates are slow relative to the iron-nitroxyl electron-electron spin-spin splitting. As temperature is increased, the relaxation rates for the Fe(III) become comparable to and then greater than the spin-spin splitting, which collapses the splitting in the continuous wave EPR spectra and causes an increase and then a decrease in the nitroxyl electron spin echo decay rate. Throughout the temperature range examined, interaction with the Fe(III) increases the spin lattice relaxation rate (1/T1) for the nitroxyl. The measured relaxation times for the Fe(III) were used to analyze the temperature-dependent changes in the spin echo decays and in the saturation recovery (T1) data for the interacting nitroxyl and to determine the interspin distance, r. The values of r for three spin-labeled methemoglobins were between 15 and 15.5 A, with good agreement between values obtained by electron spin echo and saturation recovery. Analysis of the nitroxyl spin echo and saturation recovery data also provides values of the iron relaxation rates at temperatures where the iron relaxation rates are too fast to measure directly by saturation recovery or electron spin echo spectroscopy. These results demonstrate the power of using time-domain EPR measurements to probe the distance between a slowly relaxing spin and a relatively rapidly relaxing metal in a protein.

Cyclic N-Oxides↗
PubMed1974

Investigation of very slowly tumbling spin labels by nonlinear spin response techniques: theory and experiment for stationary electron electron double resonance.

The investigation of very slowly tumbling spin labels by nonlinear electron spin response techniques is discussed. Such techniques permit characterization of rotational processes with correlation times from 10(-3) to 10(-7) sec even though the linear spin response (ESR) technique is insensitive to motion in this region. Nonlinear techniques fall into two categories: (a) Techniques (referred to as passage techniques) in which the distribution of saturation throughout the spin system is determined both by the applied magnetic field modulation of the resonance condition and by the modulation of the resonance frequency induced by the molecular motion. The time dependence of this distribution produces phase and amplitude changes in the observed signals. (b) Techniques that measure the integral of the distribution function of the time required for saturated spin packets to move between pumped and observed portions of the spectrum [stationary and pulsed electron electron double resonance (ELDOR) techniques]. Quantitative analysis of passage ESR and stationary ELDOR techniques can be accomplished employing a density matrix treatment that explicitly includes the interaction of the spins with applied radiation and modulation fields. The effect of molecular motion inducing a random modulation of the anisotropic spin interactions can be calculated by describing the motion by the diffusion equation appropriate to the motional model assumed. For infinitesimal steps the eigen-functions of the diffusion operator are known analytically, while for random motion of arbitrary step size they are determined by diagonalizing the transition matrix appropriate for the step model used. The present communication reports investigation of the rotational diffusion of the spin label probes 2,2,6,6-tetramethyl-4-piperidinol-1-oxyl and 17beta-hydroxy-4',4'-dimethylspiro-[5alpha-androstane-3,2'-oxazolidin]-3'-oxyl in sec-butylbenzene. Experimental spectra are compared with computer simulations of spectra carried out for isotropic Brownian (limit of infinitesimal step size) and free diffusion (arbitrary step size) models.

Benzene Derivatives↗
PubMedDate not supplied

[Structural studies of human IgA1 and IgA2 immunoglobulins labeled with two different spin labels].

The spin-label method was used for structural study of different subclasses of human immunoglobulin A. The spin label was incorporated into the protein part, as well as into carbohydrates of the IgA molecules. Well resolved outer wide extrema were characteristic of the ESR spectra of IgA spin-labeled at the protein moiety. ESR spectra of IgA tagged at carbohydrates reflected moderately immobilized rotation of the spin label. Dependencies of the parameters of ESR spectra of spin-labeled IgA1 and IgA2 upon viscosity at constant temperature have been investigated and a quantitative analysis of the isotherms was carried out. Spin-labeled oligosaccharide chains of IgA2 possessed great freedom of rotation. At least some of IgA1 oligosaccharides were closely attached to the protein moiety. Both proteins under study have shown flexible structure. The Fc fragment of IgA1 molecule appeared to have a rigid structure.

Electron Spin Resonance Spectroscopy↗
PubMed2006

Ultra-stable temperature control in EPR experiments: thermodynamics of gel-to-liquid phase transition in spin-labeled phospholipid bilayers and bilayer perturbations by spin labels.

An ultra-stable variable temperature accessory for EPR experiments with biological samples has been designed and tested. The accessory is comprised from a digitally controlled circulator bath that pumps fluid through high-efficiency aluminum radiators attached to an EPR resonator of a commercial X-band EPR spectrometer. Temperature stability of this new accessory after a 15 min re-equilibration is at least +/-0.007 K. For a standard 1-cm-long capillary sample arranged inside an EPR tube filled with silicon oil, the temperature variations do not exceed +/-0.033 K over the sample temperature range from 283 to 333 K. This new accessory has been tested by carrying out a comparative spin-labeling EPR and differential scanning calorimetry (DSC) study of the gel-to-liquid phase transition in multilamellar vesicles (MLV) composed of a synthetic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC). We demonstrate that the gel-to-liquid phase transition temperatures of MLV DMPC measured by EPR and DSC agree within +/-0.02 K experimental error even though the sample for EPR study was labeled with 1 mol% of 5PC (1-palmitoyl-2-stearoyl-(5-doxyl)-sn-glycero-3 phosphocholine). Cooperative unit number measured by EPR, N=676+/-36, was almost 50% higher than that obtained from DSC (N=458+/-18). These high values of N indicate that (i) the lipid domains should include at least several spin-labeled lipid molecules and (ii) the spin-probe 5PC molecules are not excluded into domains that are different from the bulk lipid phase as was speculated earlier. Overall, our data provide DSC and EPR evidence that in studies of the gel-to-liquid phase transition, the effect of bilayer perturbation by spin-labeled lipids is negligible and therefore thermodynamic parameters of the phase transition can be accurately measured by spin-labeling EPR. This might serve as an indication when spin-labeled molecules with structures similar to those of lipids are introduced at low concentrations, they are easily accommodated by fluid phospholipid bilayers without significant losses of the lipid cooperativity.

Electron Spin Resonance Spectroscopy↗
PubMed1992

Effects of polar carotenoids on dimyristoylphosphatidylcholine membranes: a spin-label study.

Spin labeling methods were used to study the structure and dynamic properties of dimyristoylphosphatidylcholine (DMPC) membranes as a function of temperature and the mole fraction of polar carotenoids. The results in fluid phase membranes are as follows: (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin, increase order, decrease motional freedom and decrease the flexibility gradient of alkyl chains of lipids, as was shown with stearic acid spin labels. The activation energy of rotational diffusion of the 16-doxylstearic acid spin label is about 35% less in the presence of 10 mol% of zeaxanthin. (2) Carotenoids increase the mobility of the polar headgroups of DMPC and increase water accessibility in that region of membrane, as was shown with tempocholine phosphatidic acid ester. (3) Rigid and highly anisotropic molecules dissolved in the DMPC membrane exhibit a bigger order of motion in the presence of polar carotenoids as was shown with cholestane spin label (CSL) and androstane spin label (ASL). Carotenoids decrease the rate of reorientational motion of CSL and do not influence the rate of ASL, probably due to the lack of the isooctyl side chain. The abrupt changes of spin label motion observed at the main phase transition of the DMPC bilayer are broadened and disappear at the presence of 10 mol% of carotenoids. In gel phase membranes, polar carotenoids increase motional freedom of most of the spin labels employed showing a regulatory effect of carotenoids on membrane fluidity. Our results support the hypothesis of Rohmer, M., Bouvier, P. and Ourisson, G. (1979) Proc. Natl. Acad. Sci. USA 76, 847-851, that carotenoids regulate the membrane fluidity in Procaryota as cholesterol does in Eucaryota. A model is proposed to explain these results in which intercalation of the rigid rod-like polar carotenoid molecules into the membrane enhances extended trans-conformation of the alkyl chains, decreases free space in the bilayer center, separate the phosphatidylcholine headgroups and decreases interaction between them.

Affinity Labels↗
PubMedDate not supplied

[Determination of the distance between spin labels and the paramagnetic center in spin-labeled proteins according to parameters of the saturation curves of the label EPR spectra at 77 degrees K].

A new method of estimation of the distance between spins of the spin-label and paramagnetic center is suggested. Method is based on the quantitative analys of saturation curve of spin-label EPR spectra at 77 degrees K. New approaches have been tested using haemoglobin labeled on SH-groups with various iminoxyl radicals. Values of the distances between labels and haem estimated from the saturation curve parameters and by current methods and values of distances estimated from X-ray data are in good agreement. In the case of rapid spin relaxation of he paramagnetic center, the new method allows one to determine a farther distance. Results of the present work make it possible to investigat by the spin-label technique the structure of haem-containing propeins and the structure of other proteins with known spin relaxation time of paramagnetic center.

Chemical Phenomena↗
PubMed1994

Effects of probucol on phase transition and fluidity of phosphatidylcholine membranes: a spin label study.

Spin labeling methods were applied to study the structure and dynamics of phosphatidylcholine membranes as a function of temperature and the mole fraction of probucol. Multilamellar liposomes made of dimyristoylphosphatidyclcholine, dipalmitoylphosphatidylcholine both saturated, and egg yolk phosphatidylcholine, an unsaturated membrane, were used. In fluid phase membranes probucol was found to increase the order and decrease the motional freedom of alkyl chains of lipids as shown with stearic acid spin labels. The effect of probucol on order and motional freedom is more pronounced in the membrane center (16-doxylstearic acid spin label position) than in the near polar headgroup region (5-doxylstearic acid spin label position). The presence of unsaturation in alkyl chains significantly decreased the ordering effect of probucol. The main phase transition temperature of saturated bilayers was lowered by 2 degrees C in the presence of 3 mol% of probucol and significantly broadened at higher concentrations as measured with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) partitioning. Also, pretransition was no longer observed in the presence of probucol. In gel phase membranes, the effect of probucol was complex. Close to the main phase transition the motion of alkyl chains was increased, showing a regulatory effect of probucol on membrane fluidity. It is proposed that probucol is located in the membrane center as opposed to vitamin E, which locates its phenolic -OH group at the membrane surface; therefore, it inhibits lipid peroxidation in this region which is less accessible to vitamin E.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1988

Interaction of spin-labeled tryptamine with monoamine oxidase: probing the microenvironment of the active site by spin probe-spin label techniques.

The spin-labeled substrate, tryptamine, was used as a structural probe of the active site of bovine liver monoamine oxidase B (amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4). When the reaction was monitored by electron spin resonance (ESR), line broadening effects indicative of binding with an apparent relation to substrate specificity of the highly purified enzyme were observed. The spectrum indicated that the bound tryptamine was 'partially immobilized' with a dissociation constant of 39 microM and 2.2 mol bound per enzyme dimer. The correlation time, reflecting the environment of the tryptamine binding site, was determined to be 6.2 ns. The topology of the active site was investigated by using dual spin-label methodology in which the spin-labeled substrate, tryptamine, and the 15N-substituted and deuterated maleimide spin label covalently bound to the essential sulfhydryl groups were used. The ESR spectral data suggested that the essential sulfhydryl groups are at least 14 A away from the tryptamine-binding site. The environment surrounding both spin-labeled substrates, tryptamine and [2H,15N]MSL, and the motional properties of the enzyme are discussed.

Binding Sites↗
PubMed1993

Measurement of rat brain perfusion by NMR using spin labeling of arterial water: in vivo determination of the degree of spin labeling.

In vivo NMR experiments are performed to determine the degree of spin labeling for measurement of tissue perfusion by NMR using spin labeling of arterial water by adiabatic fast passage. Arterial water spins are labeled using flow in the presence of a field gradient and B1 irradiation to fulfill the conditions for adiabatic fast passage spin inversion. It is demonstrated that the NMR-measured tissue perfusion is not affected by changing the degree of spin labeling as long as the degree of spin labeling is determined and accounted for according to the model used for calculating perfusion. By measuring the degree of spin labeling with different arterial blood flow velocities induced by different arterial pCO2, it is also demonstrated that, when spin labeling is carried out by adiabatic fast passage, the degree of spin labeling is not affected by changes in arterial blood flow velocity over a broad range.

Animals↗
PubMed1988

Interaction of spin-labeled tryptophan with sickle hemoglobin: probing the microenvironment of the contact sites by spin-probe--spin-label techniques.

The spin-labeled tryptophan was used as a structural probe of hemoglobin contact sites. The ESR spectral data indicated that the probe exhibits weak binding to hemoglobin with a dissociation constant of 3.2.10(-5) and 4.0 mol bound per hemoglobin tetramer. The spectrum suggested that the bound tryptophan was 'partially immobilized' with a correlation time reflecting the environment of the tryptophan binding site of 8.2 ns. The topology of the contact sites was investigated by using dual spin-label methodology in which spin-labeled tryptophan and (2H,15N) substituted and deuterated maleimide spin label [2H-15N]MSL covalently-bound to Cys-beta 93 residue were used. The ESR spectral data suggested that the tryptophan binding sites were located within 8-10 A of the nitroxide free radical of spin-labeled hemoglobin. The environment of the contact sites is discussed.

Cysteine↗
PubMed1991

Two dimensional diffusion of small molecules on protein surfaces: an EPR study of the restricted translational diffusion of protein-bound spin labels.

Heisenberg spin exchange rates and dipole-dipole spin lattice relaxation rates for deuterated 14N- and 15N-spin labels bound selectively to the histidine His15 and to the lysines Lys13, 96, 97 of the lysozyme molecule have been determined with the aid of electron spin resonance spectroscopy. The results can be interpreted in terms of a two dimensional translational diffusion of the nitroxide tips of the spin labels along the protein surface within restricted surface areas. The spin labels are regarded as models for long amino acid side chains and as probes for the dynamics of protein and water in the vicinity of the protein surface. The translational diffusion coefficient DII is reduced by a factor of between six and thirty compared to the value for T = 295 K is given by (1.3 +/- 0.6) x 10 -10m2s-1 greater than or equal to DII greater than or equal to (2.4 +/- 0.3) x 10-11M2s-1.

Amino Acid Sequence↗
PubMed1982

Oxygen transport parameter in membranes as deduced by saturation recovery measurements of spin-lattice relaxation times of spin labels.

Spin-lattice relaxation time (T1) measurements of nitroxide radical spin labels in membranes have been made by using the saturation-recovery technique. Stearic acid and sterol-type labels were used as probes of dimyristoylphosphatidylcholine liposomes from 0 degrees C to 36 degrees C. In the absence of oxygen, the range of variation of T1 over all samples and conditions is about a factor of 3. Heisenberg exchange between oxygen and spin labels is an effective T1 mechanism for the spin labels. The full range of variation of T1 in the presence of air is about a factor of 100. It is suggested that the oxygen transport parameter W = T1(-1) (air) - T1(-1) (N2) is a useful new monitor of membrane fluidity that reports on translational diffusion of small molecules. The values of W change at the prephase and main phase transitions and vary in complex ways. Arguments are advanced that the data are indicative of anisotropic translational diffusion of oxygen.

Diffusion↗
PubMed2001

Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location.

Phase memory relaxation times (T(M) or T(2)) of spin labels in human carbonic anhydrase II (HCA II) are reported. Spin labels (N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide, IPSL) were introduced at cysteines, by site-directed mutagenesis at seven different positions in the protein. By two pulse electron paramagnetic resonance (EPR), electron spin echo decays at 45 K are measured and fitted by stretched exponentials, resulting in relaxation parameters T(M) and x. T(M) values of seven positions are between 1.6 micros for the most buried residue (L79C) and 4.7 micros for a residue at the protein surface (W245C). In deuteriated buffer, longer T(M) are found for all but the most buried residues (L79C and W97C), and electron spin echo envelop modulation (ESEEM) of deuterium nuclei is observed. Different deuterium ESEEM patterns for W95C and W16C (surface residue) indicate differences in the local water concentration, or accessibility, of the spin label by deuterium. We propose T(M) as a parameter to determine the spin label location in proteins. Furthermore, these systems are interesting for studying the pertaining relaxation mechanism.

Carbonic Anhydrase II↗
PubMed1984

Dipsticking the major groove of DNA with enzymatically incorporated spin-labeled deoxyuridines by electron spin resonance spectroscopy.

Site-specifically spin-labeled deoxyuridine triphosphates with tethers of different lengths were synthesized and then enzymatically incorporated with terminal transferase to form a spin-labeled poly(dT) copolymer. The spin-labeled copolymers were annealed with poly(dA) to form a duplex, which was analyzed by electron spin resonance spectroscopy in a solution of low ionic strength. The spin labels are attached in position 5 of the deoxyuridine and protrude into the major groove. Based on the correlation between tether length of the spin label and the electron spin resonance lineshape, we show that the depth of the major groove of a DNA in its B-form is about 8 A in solution, which is in good agreement with X-ray fiber studies. We also conclude, based on electron spin resonance lineshape simulation data, that the correlation time of the bases in a DNA duplex is of the order of nanoseconds.

DNA↗
PubMed1990

Effect of ADP on the orientation of spin-labeled myosin heads in muscle fibers: a high-resolution study with deuterated spin labels.

We have used electron paramagnetic resonance (EPR) to determine the effects of ADP on the orientational distribution of nitroxide spin labels attached to myosin heads in skinned rabbit psoas muscle fibers. To maximize the specificity of labeling, we spin-labeled isolated myosin heads (subfragment 1) on a single reactive thiol (SH1) and diffused them into unlabeled muscle fibers. To maximize spectral and orientational resolution, we used perdeuterated spin labels, 2H-MSL and 2H-IASL, eliminating superhyperfine broadening and thus narrowing the line widths. Two different spin labels were used, with different orientation relative to the myosin head, to ensure that the results are not affected by unfavorable probe orientation. In rigor, a very narrow three-line spectrum was observed for both spin labels, indicating a narrow orientational distribution, as reported previously (Thomas & Cooke, 1980). ADP induced very slight changes in the spectrum, corresponding to very slight (but significant) changes in the orientational distribution. These changes were quantified by a digital analysis of the spectra, using a two-step simplex fitting procedure (Fajer et al., 1990). First, the magnetic tensor values and line widths were determined by fitting the spectrum of a randomly oriented sample. Then the spectrum of oriented fibers was fit to a model by assuming a Gaussian distribution of the tilt angle (theta) and twist angle (phi) of the nitroxide principal axes relative to the fiber axis. A single-Gaussian distribution resulted in inadequate fits, but a two-component model gave excellent results. ADP induces a small (less than 5 degrees) rotation of the major components for both spin labels, along with a similarly small increase of disorder about the average positions.

Actin Cytoskeleton↗