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PubMed1982

Spin-label saturation-transfer electron spin resonance detection of transient association of rhodopsin in reconstituted membranes.

Rotational diffusion of rhodopsin in reconstituted membranes of phosphatidylcholines of various alkyl chain lengths has been measured by using saturation-transfer electron spin resonance spectroscopy as a function of temperature and lipid/rhodopsin mole ratio. For dipalmitoyl-phosphatidylcholine, the rotational correlation time is 20 microseconds at physiological concentration, the same as in rod outer segment (ros) membranes. Dilution reduces the time to 10 microseconds, a value that is ascribed to well-dispersed monomeric rhodopsin. Use of phospholipids with longer or shorter chains results in sharply increased rotational correlation times. It is concluded that rhodopsin molecules are transiently associated in both reconstituted and ros membranes and that the nature of the association is determined by lipid type and composition.

Animals↗
PubMedDate not supplied

[Study of yeast phosphoglycerate kinase by electron paramagnetic resonance. 1. Modification of protein SH-group by spin labels. Conformational changes of enzyme].

The conditions for modifications of a single SH-group of phosphoglycerate kinase from yeast by maleimid spin label and the mercury-containing spin labels were defined. The time course of immobilization of mercury containing paramagnetic radicals was obtained. Based on changes in EPR spectra we calculated dissociation constants for complexes of phosphoglycerate kinase with 3-phosphoglycerate and sulfate ions. Distinct substances were shown to induce different changes in enzyme conformation. It was concluded that approach of enzyme domains is caused by the binding of 3-phosphoglycerate-a specific substrate of this enzyme.

Electron Spin Resonance Spectroscopy↗
PubMed1999

A novel steroidal spin label for membrane structure studies: synthesis and applications.

2,2,6,6-Tetramethyl piperidine-N-oxyl nitroxyls are known to partition between aqueous and lipid phases, thus serving as probes to study membrane dynamics. The synthesis of a novel steroidal spin label, 3alpha-hydroxycholan-24-yl-(2",2",6",6"-tetramethyl-N-oxyl)p iperidyl butan-1',4'-dioate, containing 2,2,6,6-tetramethylpiperidine-N-oxyl moiety covalently bonded to the side chain in 3,24-caprostan-diol has been described. The localization of this spin label in model biomembranes has been studied by using electron spin resonance, differential scanning calorimetry, and 1H and 31P NMR spectroscopic techniques. Its applicability in studying the phase transition properties of model membrane L-alpha-dipalmitoyl phosphatidyl choline in the presence and absence of drugs has been described by using electron spin resonance. The label has also been used to study the permeability of epinephrine into membrane. The results have shown the applicability of the spin label as a potential spin probe in the study of biomembranes.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1987

Orientation and vertical fluctuations of spin-labeled analogues of cholesterol and androstanol in phospholipid bilayers.

We have used ESR and NMR linewidth broadening by spin-labels to determine the overall orientation of spin-labeled analogues of cholesterol and androstanol in egg lecithin bilayers. While the cholesterol analogues were found to have a single orientation in each monolayer, with the acyl chain pointing towards the center of the bilayer, the androstanol analogue appeared, at least in sonicated vesicles, to experience two opposite orientations in the same monolayer, very likely with a rapid reorientation. The possibility of rapid vertical fluctuations of the sterol molecules within the phospholipid bilayer is also discussed.

Androstanols↗
PubMed1980

Spin label partitioning in lipid vesicles. A model study for drug encapsulation.

In this paper we attempt to outline some features which determine the encapsulation of small molecules into lipid vesicles. Spin labels derived from five carboxylic acids of different lengths were synthesized and incorporated in varying amounts into multilamellar and unilamellar vesicles made up of four different phosphatidylcholines. The influence on the release process of the bilayer rigidity and of the hydrophobicity of the entrapped molecule was systematically studied. The hydrophobicity is of critical importance and was estimated by measuring the partition coefficient (P) between octanol and buffer. In multilamellar vesicles, molecules characterized by extreme P values (log P less than -0.3 and log P greater than 5) can be efficiently entrapped. The rate of leakage is related to the P value according to a bell-shaped curve. Moreover, gel state of the bilayer and long acyl chains of the lipids are properties which favor a good entrapment. Small unilamellar vesicles may be formed in the presence of high concentrations of hydrophilic and lipophilic spin labels. However, the formation of unilamellar vesicles produces a significant reduction of the internal volume and of the entrapped water-soluble spin lables. High fractions of lipid-soluble spin labels can be incorporated in unilamellar vesicles but the vesicle stability is diminished.

Carboxylic Acids↗
PubMed1996

Determination of the orientation of a band 3 affinity spin-label relative to the membrane normal axis of the human erythrocyte.

The orientation of the nitroxide moiety of an isotopically substituted spin-labeled derivative of dihydrostilbenedisulfonate ([15N,2H13]-SL-H2DADS-maleimide) covalently coupled at the extracellular stilbenedisulfonate binding site of the human erythrocyte anion exchange protein, band 3, has been determined relative to the membrane normal axis of intact cells. The X-band linear electron paramagnetic resonance (EPR) spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled band 3 in intact erythrocytes oriented by flow through an EPR flat cell have been obtained for two orthogonal orientations of the sample in the DC magnetic field. Two different methods of analysis have provided very similar values for the angles alpha 1 and beta 1 which uniquely define the orientation of the nitroxide axis frame relative to the membrane normal axis. In the first approach, a variable fraction of the cells, f, were taken to be biconcave disks perfectly oriented relative to the flat cell surface with the remainder, 1-f, isotropically oriented. Simultaneous nonlinear least squares analysis of the spectra obtained at the two sample orientations yielded best fit values of f = 0.60, alpha 1 = 58 degrees, and beta 1 = 36 degrees. In the second approach, the EPR spectra of flow-oriented intact erythrocytes labeled with the fatty acid spin-label, [15N,2H12]-5-nitroxyl stearate, have been obtained at the two sample orientations. These two spectra have been used to determine a model-independent distribution of membrane normal orientations in the sample. Using this experimentally determined membrane normal orientation distribution, the EPR spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled erythrocytes were then reanalyzed to obtain a second determination of the nitroxide orientation, alpha 1 = 61 degrees and beta 1 = 37 degrees. The orientation of the nitroxide with respect to the membrane normal axis determined in the present study is nearly identical to the orientation of the nitroxide with respect to the uniaxial rotational diffusion axis, alpha = 66 degrees and beta = 34 degrees, as determined from saturation transfer EPR (ST-EPR) studies [Hustedt, E. H., & Beth, A. H. (1995) Biophys. J. 69, 1409-1423]. This result supports the conclusion that the motion observed using ST-EPR spectroscopy is, in fact, the uniaxial rotational diffusion of band 3 about the membrane normal.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1993

Oligonucleotides site-specifically spin-labeled at 5'-terminal or internucleotide linkage and their use in gene analyses.

Spin-labeled oligonucleotides (S-probes) were synthesized and examined as DNA probes to monitor hybrid formation. TEMPO was introduced either at the internucleotide linkage of 5'-terminus (Type 1) or at the 5'-terminal hydroxyl group (Type 2) and both types of S-probes were used in this study. The presence of target DNA was detected in solution by EPR spectroscopy for both types of S-probes. Hybridization of the S-probes resulted in notable broadening of EPR line width, accompanied by a decrease in the EPR signal height ratio for I(-1)/I(0).I(-1)/I(0) of S-probes having no spacer between oligonucleotide and TEMPO decreased more markedly than that of S-probes with a spacer, indicating that TEMPO should be introduced to an oligonucleotide directly to monitor hybrid formation. When M13mp8 single-stranded DNA with or without an EcoRI recognition site was selected as a target DNA, hybrid formation was detected only for DNA containing EcoRI site in solution using spin-labeled oligonucleotides.

Bacteriophage M13↗
PubMed1993

Lipid chain motion in an interdigitated gel phase: conventional and saturation transfer ESR of spin-labeled lipids in dipalmitoylphosphatidylcholine-glycerol dispersions.

The lipid chain dynamics in the interdigitated gel phase of dipalmitoylphosphatidylcholine (DPPC) dispersed in glycerol and in the fully hydrated noninterdigitated gel phase in aqueous buffer were compared by using conventional and saturation transfer electron spin resonance (ESR) spectroscopy. Twelve different positional isomers of phosphatidylcholine spin-labeled in the sn-2 chain were used to characterize the chain motion. The outer hyperfine splittings of the conventional ESR spectra and the line height ratios at the diagnostic spectral positions in the saturation transfer ESR spectra were taken as indices of the rotational mobility of the labeled chain segments in the gel phase (0-40 degrees C). The conventional spin label ESR spectra revealed a gradient of increasing mobility on proceeding down the chain toward the terminal methyl end in the fully hydrated DPPC gel phase bilayer structure. This gradient was absent in the interdigitated gel phase, i.e., the rotational mobility throughout the length of the lipid chain was comparable to that near the polar interface, on the conventional ESR time scale. Values of the outer hyperfine splitting for spin labels at the 5- and 14-C atom positions in the chain were 65.5 and 61.0 G in buffer, respectively, and 67.0 G for both positions in glycerol, at 0 degrees C. At 35 degrees C, still in the gel phase, these differences between the two systems were much greater. Saturation transfer ESR measurements revealed that the motion throughout the chain was restricted on the microsecond time scale in the interdigitated phase.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1981

Use of spin label and the flow-induced ESR spectral difference for studying erythrocyte deformation.

ESR spin-labeling method is expanded to measure the macroscopic visco-elastic properties of erythrocytes. A suspension of erythrocytes with an incorporated fatty acid spin label was forced to flow through a flat ESR sample cells, and the ESR spectral change caused by the shear flow was utilized to assess the cell deformability. Chemical cross-linking or heat denaturation of membrane proteins to make the cells less deformable without any morphological change was found to reduce the relative spectral difference (delta h/h). This result indicates that the spectral difference is related to the cell deformation that accompanies the orientation of the cells in the shear flow. In addition, the average decay time (tau av) for the spectral difference observed when the flow was abruptly interrupted became shorter with an increase in the degree of cross-linking or heat-denaturation abruptly interrupted became shorter with an increase in the degree of cross-linking or heat-denaturation at 49 degrees C. Since the observed tau av is much shorter than the expected rotational correlation time for the erythrocyte, the decay is attributed to the deformation recovery process. It is demonstrated that the measurements of both delta h/h and tau av by ESR spectroscopy give qualitative information on the viscosity and the elasticity of the cell membrane system.

Blood Viscosity↗
PubMed2004

Librational motion of spin-labeled lipids in high-cholesterol containing membranes from echo-detected EPR spectra.

Two-pulse, echo-detected (ED) electron paramagnetic resonance (EPR) spectroscopy was used to study the librational motions of spin-labeled lipids in membranes of dipalmitoylphosphatidylcholine + 50 mol % cholesterol. The temperature dependence, over the range 77-240 K, and the dependence on position of spin-labeling in the sn-2 chain (n=5, 7, 10, 12, and 14) of the phospholipid, were characterized in detail. The experimental ED-spectra were corrected for instantaneous spin diffusion arising from static spin-spin interactions, by using spectra recorded at 77 K, where motional contributions are negligible. Simulations according to a model of rapid, small-amplitude librations about an axis whose direction is randomly distributed are able to describe the experimental spectra. Calibrations, in terms of the amplitude-correlation time product, alpha2tauc, were constructed for diagnostic spectral line-height ratios at different echo delay times, and for relaxation spectra obtained from the ratio of ED-spectra recorded at two different echo delays. The librational amplitude, alpha2, was determined for a spin label at the 14-C position of the lipid chain from the partially motionally averaged hyperfine splitting in the conventional EPR spectra. The librational correlation time, tauc, which is deduced from combination of the conventional and ED-EPR results, lies in the subnanosecond regime and depends only weakly on temperature. The temperature dependence of the ED-EPR spectra arises mainly from an increase in librational amplitude with increasing temperature, and position down the lipid chain. A gradual transition takes place at higher temperatures, from a situation in which segmental torsional librations are cumulative, i.e., the contributions of the individual segments add up progressively upon going down the chain, to one of concerted motion only weakly dependent on chain position. Such librational motions are important for glass-like states and are generally relevant to high lipid packing densities, e.g., in cholesterol-containing raft domains and condensed complexes.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1975

[Study of the interaction of human serum albumin with penicillins by means of spin labels].

Interaction of 8 penicillin preparations with human serum albumin was studied with the spin-labels method and a probe. Correlation between the binding level of penicillins with human serum albumin and their effect on the spectrum of EPR of the spin-label attached to albumin was observed only with the use of a hydrophobic probe (radical III). The covalent attached marks and the hydrophobic probe may be used for rapid orienting estimation of pencillin interaction with albumin.

Ampicillin↗
PubMed1993

Spin label saturation transfer EPR determinations of the stoichiometry and selectivity of lipid-protein interactions in the gel phase.

Lipid-protein interactions with the myelin proteolipid protein incorporated in the gel phase of dimyristoylphosphatidylcholine bilayers have been studied by saturation transfer EPR spectroscopy of spin-labelled phospholipids. The integrated intensities of the saturation transfer EPR spectra from spin-labelled phosphatidylcholine are linearly dependent on the protein/lipid ratio, and correspond to a fixed stoichiometry of approximately 11 lipids per monomer associated with the protein in the gel phase. The normalized saturation transfer intensities of spin-labelled phosphatidic acid, on the other hand, display a non-linear dependence on the protein/lipid ratio that can be described well by a selectivity for interaction with the protein in the gel phase with an average association constant relative to phosphatidylcholine of approx. 5.2. These values for the stoichiometry and selectivity of lipid-protein interaction in the lipid gel phase obtained from saturation transfer EPR spectroscopy are comparable to those found previously in fluid phase lipids by conventional EPR spectroscopy.

Dimyristoylphosphatidylcholine↗
PubMed1982

Spin label studies on osmotically-induced changes in the aqueous cytoplasm of Phaeodactylum tricornutum.

The effects of hyperosmotic stress and adaption on the aqueous cytoplasm of Phaeodactylum tricornutum have been studied with spin labels using 0.2M external Ni2+ to obtain spectra solely from labels within the cells. From partitioning of the TEMPO spin label between the internal aqueous phase and the membrane it is found that the internal volume of the cells decreased by approx. 50-60% in media of high osmotic strength (1.9 osmol/l). During the accumulation of proline in the cells (8.8 mg/ml packed cells) on incubation in the medium of high osmolarity for 3 days, the recovery of the volume was 80%. Further addition of proline to the medium resulted in an increase in the proline concentration in the cells (12.2 mg/ml packed cells) and a recovery in volume of 90%. Cells incubated in the absence of any nitrogen source showed very little recovery and were in a stressed state even in the absence of an osmotic gradient. From the rotational correlation times of the TEMPONE spin label it was found that the effective microviscosity in the cytoplasm of normal cells (approx. 3-8 cP) was considerably higher than that of the external medium (1 cP) and increased 1.5-2-fold under high osmotic stress (1.9 osmol/l). Adaption during the accumulation of proline only decreased the effective microviscosity by approx. 50% of the stressed-induced increase, a considerably smaller recovery than that of the cell volume.

Acclimatization↗
PubMed1978

Spin-label studies of the sulfhydryl environment in bovine plasma albumin. 1. The N--F transition and acid expansion.

The environment of the sulfhydryl group in plasma albumin was previously characterized by employing spin-labels of varying chain lengths (Hull, H. H., Chang, R., & Kaplan, L. J. (1975) Biochim. Biophys. Acta 400, 132). It was established that the sulfhydryl is in a crevice approximately 10 A deep but this crevice was not identified further. We now report the results of titrating albumin through the acidic conformational transitions while monitoring the electron-spin resonance of the bound nitroxide. With short spin-labels a general change is observed as the pH is lowered but the N--F transition is not discernible. However, with a spin label previously shown to project to the lip on the crevice a clear N--F transition as well as the subsequent acid expansion are observed. These results indicate that the sulfhydryl is in the crevice, formed by the domains of albumin, which opens during the N--F transition. Further results indicate that bound fatty acids do not influence the integrity of the sulfhydryl environment at neutral pH.

Chemical Phenomena↗
PubMed1975

The orientation of cytochrome c oxidase in coupled phospholipid membrane vesicles--a spin-label study.

Cytochrome oxidase, an enzyme containing six different subunits, has been shown to span the inner mitochrondrial membrane. The arrangement of the subunits within the membrane is unknown. Wh have specifically labeled the 25 000 molecular weight subunit with a spin-label derivative of N-ethylmaleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (NEM-SL(5)). NEM-SL(5)-lebeled cytochrome oxidase can be incorporated into phospholipid membranes to form coupled vesicles of the Hinkle, Kim & Racker ((1972) Jriol. Chem; 247, 1338-1399) type. The resonance spectrum of NEM-SL(5) is similar in both soluble and vesicular cytochrome oxidase. Since ascorbate has been shown to reduce only spin label that is exposed to the exterior surface of a closed vesicle, we have used ascorbate to determine the NEM-SL(5)-binding site in the coupled vesicles; NEM-SL(5)-labeled cytochrome oxidase vesicles are reduced by 10 mM ascorbate with tau 1/2 of 1 min at 22 degrees C; The rate of reduction is relatively independent of temperature. We conclude that (1) cytochrome oxidase is unidirectionally or preferentially oriented in the vesicle membrane, and (2) the NEM-SL(5)-binding site on the 25 000 molecular weight subunit is exposed to the external aqueous medium.

Animals↗
PubMed1986

Investigation of membrane proteins in rat erythrocytes in spontaneous hypertension by means of spin-label technique.

It has been suggested that alterations in cell membrane proteins may play a role in changes of erythrocyte membrane structure and function in hypertension. In order to characterize the structure of membrane proteins of erythrocytes from spontaneously hypertensive rats (SHR) the spin-label technique with a maleimide spin-label was used. A significant difference was observed in the characteristic electron-spin resonance (e.s.r.) spectrum of the label between samples from normotensive rats and SHR. The difference was eliminated and the spectrum significantly changed after treatment of the labelled membrane with EDTA followed by washing out the EDTA extracts, whereas the same treatment with EDTA without the following washing had no effect on the e.s.r. spectrum. It is concluded that the EDTA extracts different substances in the different rat groups. The spin-label technique is a useful method for distinguishing cell membrane properties in SHR and normotensive rats.

Animals↗
PubMed1982

Propranolol-induced structural changes in human erythrocyte ghost membranes. A spin label study.

The effect of propranolol on the structure of human erythrocyte membranes was studied using a spin labeling technique. Changes in electron spin resonance spectra of spin labeled membrane proteins were detected at concentration of the drug corresponding to its antihemolytic effect on intact erythrocytes. The character of spectral changes suggests that propranolol-induced alterations in organization of membrane proteins are connected mainly with perturbation of protein sites located on membrane surface. Propranolol also produces a decrease in order parameter of membrane lipids. The disordering effect is, however, small and detectable only at relatively high concentrations of the drug.

Electron Spin Resonance Spectroscopy↗
PubMedDate not supplied

[Study of the effect of corticosterone on erythrocyte membranes by the spin label method].

Influence of corticosterone on the hare's erythrocyte ghosts is studied by the spin-label method. There exists a correlation between the maximal variation region in the rotational diffusion "frequency" of the spin-labels, covalently linked with the membrane protein, and the hormones concentration range within which the effect of corticosterone on the functions of the erythrocyte membranes manifests itself to the greatest degree (permeability to ions, hemolytic effects, etc).

Animals↗