Water-soluble Spin Label Research Articles

The Interaction of Water-Soluble Nitroxide Radicals with Photosystem II.

In this work, we investigated the redox transients of a number of water-soluble spin labels upon their interactions with Photosystem II (PS II) core complexes isolated from spinach leaves. We have found that the reactivity of nitroxide radicals, determined by the rate of their reduction upon illumination of PS II, depends on the chemical structure of radicals and the capability of their coming close to low-potential redox centers of photoactive PS II complexes. An enhanced capability of nitroxide radicals to accept electrons from PS II correlates with their chemical structure. Nitroxide radicals NTI (2,2,5,5-tetramethyl-4-nitromethylene-3-imidazolidine-N-oxyl) and Tacet (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl-acetate), containing polar groups, appear to be most efficient acceptors of electrons donated by PS II compared to neutral (TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) or positively charged (Tamine, 4-amino-2,2,6,6-tetramethylpiperidine-l-oxyl) spin labels. We assume that enhanced reactivities of polar nitroxide radicals, NTI and Tacet, are determined (1) by their relatively high redox potentials, providing the possibility to accept electrons from PS II, and (2) by their affinities to the closest binding sites on the surface of PS II in the vicinity of the primary plastoquinone acceptor PQA (12–14 Å) or/and in the intraprotein cavity for the secondary plastoquinone PQB (~ 22 Å).

Spin-label oximetry at Q- and W-band

Spin–lattice relaxation times ( T 1s) of small water-soluble spin-labels in the aqueous phase as well as lipid-type spin-labels in membranes increase when the microwave frequency increases from 2 to 35 GHz (Hyde, et al., J. Phys. Chem. B 108 (2004) 9524–9529). The T 1s measured at W-band (94 GHz) for the water-soluble spin-labels CTPO and TEMPONE (Froncisz, et al., J. Magn. Reson. 193 (2008) 297–304) are, however, shorter than when measured at Q-band (35 GHz). In this paper, the decreasing trends at W-band have been confirmed for commonly used lipid-type spin-labels in model membranes. It is concluded that the longest values of T 1 will generally be found at Q-band, noting that long values are advantageous for measurement of bimolecular collisions with oxygen. The contribution of dissolved molecular oxygen to the relaxation rate was found to be independent of microwave frequency up to 94 GHz for lipid-type spin-labels in membranes. This contribution is expressed in terms of the oxygen transport parameter W = T 1 - 1 ( Air ) - T 1 - 1 ( N 2 ) , which is a function of both concentration and translational diffusion of oxygen in the local environment of a spin-label. The new capabilities in measurement of the oxygen transport parameter using saturation-recovery (SR) EPR at Q- and W-band have been demonstrated in saturated (DMPC) and unsaturated (POPC) lipid bilayer membranes with the use of stearic acid ( n-SASL) and phosphatidylcholine ( n-PC) spin-labels, and compared with results obtained earlier at X-band. SR EPR spin-label oximetry at Q- and W-band has the potential to be a powerful tool for studying samples of small volume, ∼30 nL. These benefits, together with other factors such as a higher resonator efficiency parameter and a new technique for canceling free induction decay signals, are discussed.

On the surface properties of oleate micelles and oleic acid/oleate vesicles studied by spin labeling

Dilute aqueous systems composed of sodium oleate micelles and sodium oleate/oleic acid vesicles were investigated as a function of pH by electron spin resonance spectroscopy with TEMPO-stearate TEMPO-stearamide as well as with a positively charged water soluble spin label, TEMPO-choline. The dynamics of the three TEMPO-spin labels were found to be sensitive to changes in the interfacial region of the aggregates as a function of pH. The results obtained are consistent with the formation of a hydrogen bond network (RCOO−↔HOOCR) at the surface of the sodium oleate/oleic acid system in the course of the transformation of micelles into the closed bilayers (vesicles). Vesicles formation below pH 10 was determined independently with a spin labeled glucose derivative.

Mixtures of semisynthetic species of cerebroside sulfate with dipalmitoyl phosphatidylcholine. Thermotropic phase behavior and permeability

The phase behavior of mixtures of dipalmitoyl phosphatidylcholine (DPPC) with semisynthetic species of cerebroside sulfate (CBS) containing palmitic acid (C16:0-CBS) or lignoceric acid (C24:0-CBS) in 0.1 M KCl was studied using differential scanning calorimetry. DPPC and C16:0-CBS were miscible in all proportions in the gel phase above 10 mol% CBS and in the liquid-crystalline phase. However, C24:0-CBS was less miscible with DPPC over a wide concentration range in the gel phase. At high CBS concentrations it was probably also not entirely miscible with DPPC in the liquid-crystalline phase. Small amounts of both species of CBS lowered the transition temperature and enthalpy of DPPC, suggesting that they are more soluble in the liquid-crystalline phase of DPPC than the gel phase. The transition temperature at higher CBS concentrations was also less than expected, especially after cycling through the phase transition in the case of C24:0-CBS, suggesting that mixing with DPPC inhibited the intermolecular hydrogen bonding interactions and dehydration of CBS. In C24:0-CBS-DPPC mixtures several populations were present over a wide compositional range, including two solid-solid solutions of fixed composition. At high C24:0-CBS concentrations some C24:0-CBS also phase separated out of the mixture. Structural considerations suggested that the C24:0-CBS which is mixed with DPPC must be interdigitated into the DPPC bilayer. Other populations that are present may have a different structural organization. A fatty acid spin label in these mixtures was a little less ordered than in either lipid by itself. The permeability of these lipids, as well as the two asymmetric species 1-stearoyl-2-caproyl phosphatidylcholine and 1-stearoyl-2-myristoyl phosphatidylcholine (18:10PC and 18:14PC), to a water-soluble spin label tempocholine chloride was also measured. The studies with 18:10PC and 18:14PC indicated that both triple-chain mixed interdigitated bilayers and double-chain partially interdigitated bilayers can trap water-soluble substances and have low permeability. Both species of CBS could also entrap the spin label and had low permeability at 4 degrees C. However, they rapidly lost the entrapped compound when they transformed into their stable dehydrated phases or into the liquid-crystalline phase. Mixing with DPPC prevented both of these losses. These studies supported the conclusion that a significant amount of the CBS was mixed with the DPPC and that this mixing prevented the dehydration changes which CBS undergoes by itself.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytotoxicity of commonly used nitroxide radical spin probes

Since nitroxide radical spin probes are used frequently to test biophysical properties of cells, their use should be restricted to conditions that do not perturb normal cell growth and viability. Eight commonly used nitroxide radical spin probes have been tested for their effects on the survival of CHO cells. These include water-soluble spin probes Tempol, Tempamine, CTPO, CTPC and 4-maleimido-Tempo, and lipid soluble spin probes 5-Doxyl-, 12-Doxyl-, and 16-Doxylstearates. With the exception of 4-maleimido-Tempo, none of the water soluble spin labels inhibited cell survival at concentrations as high as 1 mM. At concentrations of 75 μM and higher, 4-maleimido-Tempo inhibited cell survival in a dose dependent manner. At concentrations commonly used for spin labeling of cells (30–50 μM) none of the lipid soluble spin probes tested was cytotoxic. At 100 μM only 5-Doxylstearate inhibited cell survival, whereas 12-Doxylstearate and 16-Doxylstearate had no effect.

Spin label study of local anesthetic-lipid membrane interactions. Phase separation of the uncharged from and bilayer micellization by the charged form of tetracaine

The interaction between tetracaine and egg phosphatidylcholine (egg PC) multibilayers was examined. ESR spectra of an ester spin label indicate that at low uncharged anesthetic: lipid ratios, membrane organization decreases. At higher ratios, saturation and phase separation occur, as suggested by a second spectral component which appears when the water solubility of tetracaine is reached. However, experiments with the drug in the absence and in the presence of membranes, making use of a phospholipid spin label, suggest that the new phase does not consist of solid tetracaine alone. Location of the new phase in the membrane would require a change in partition coefficient, while its location outside would imply a mechanism whereby the anesthetic would come off the membrane as aggregate containing spin probe and phospholipid. Charged tetracaine forms micelles which disrupt-unilamellar egg PC vesicles (Fernandez, M.S. (1981) Biochim. Biophys. Acta 646, 27–30). Micellar tetracaine added to bilayers containing a PC spin probe changes the spectrum from one typical of a bilayer into one typical of micelles, indicating the formation of a tetracaine-egg PC mixed micelle. The effect is reversible upon dilution to concentrations below the critical micelle concentration of tetracaine. When membranes are prepared in the presence of a water-soluble spin label, TEMPOcholine, ascorbate destroys the signal of untrapped label; when mixed phospholipid-tetracaine are formed by addition of micellar tetracaine, this leads to a complete loss of the ESR signal. High drug concentrations are often used for anesthesia and could be related to morphological nerve damage caused by large doses of anesthetics.

Increase in vesicle permeability mediated by myelin basic protein: effect of phosphorylation of basic protein

The two most basic charge isomers of myelin basic protein (BP), components 1 and 2 (C1 and C2), which presumably differ in the degree of deamidation, were purified from bovine BP by cation-exchange chromatography. Two additional specific types of posttranslational modifications were introduced into the purified isomers: (1) C-terminal arginine deficient derivatives of C1 and C2 were prepared by incubating the isomers with a carboxypeptidase, and (2) phosphorylated derivatives of C1 (1.6 and 1.7 mol of phosphate/mol of protein) were prepared by incubating C1 with the protein kinase from rabbit muscle. The ability of these charge isomers to increase the permeability of multilamellar vesicles composed of phosphatidylserine/phosphatidylcholine (1:11.5 w/w) and sphingomyelin/cholesterol/phosphatidic acid (1:1:0.2 w/w/w) was measured by monitoring the release of a water-soluble spin-label (tempocholine chloride) from the vesicles. The increase in vesicle permeability caused by BP was taken as a measure of the degree of perturbation of the bilayer by the protein, most likely by penetration partly into the bilayer. All classes of charge isomers (naturally occurring or generated in vitro) were more effective at increasing vesicle permeability than was poly(L-lysine), a polycation that only interacts electrostatically with the bilayer. Although C1 and C2 and their C-terminal-deficient derivatives did not differ in the amount of marker released, the phosphorylated derivative of C1 caused a smaller increase in vesicle permeability than did the other isomers, suggesting that phosphorylation had altered the ability of the protein to perturb the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Immune lysis of lipid vesicles containing myelin basic protein or glycolipid antigens by multiple sclerosis and normal sera

We have compared the reactivity of sera from 34 multiple sclerosis (MS) patients and 32 normal (N) individuals with lipid vesicles containing myelin basic protein (BP) and several glycolipids reconstituted into a membrane environment. The ability of the sera to cause complement-mediated lysis of lipid vesicles containing these antigens was determined by measuring the release of a water-soluble spin label, tempocholine chloride, from the height of its electron spin resonance spectrum. Only 4 MS sera caused lysis of BP-containing vesicles which was comparable to that produced by specific antibody to BP. A number of both MS and N sera caused significant lysis of vesicles containing G m1 ganglioside or digalactosyldiglyceride. A few MS and N sera also caused significant lysis of vesicles containing G m2 , G t1 and G d1a gangliosides. However, in no case was there a statistically significant difference between the mean lysis produced by MS and N sera. There was some overlap between the specific MS and N sera reactive to vesicles containing BP, G m1 , G m2 , and DGDG while a completely different group of MS and N sera were reactive to G t1 and G d1a gangliosides. This suggested that there was either antigenic cross reactivity between the two groups of glycolipids or two different origins of the immune response to the two groups of antigens. It was concluded that antibody-dependent complement fixation by these particular antigens, in the kind of lipid environment used, is not characteristic of or specific to MS.

Spin membrane immunoassay for use in meningococcal serology.

A modified and improved spin membrane immunoassay has been developed for detecting complement-activating antibodies to Neisseria meningitidis capsular polysaccharide antigens. The polysaccharides were incorporated in the membranes of large unilamellar vesicles prepared by the reverse-phase evaporation method and filled with the water-soluble spin label tempocholine chloride. Upon addition of group-specific antisera and complement, the lipid membrane was damaged and the spin label leaked out. This process was monitored by electron spin resonance spectroscopy. A satisfactory assay was developed for polysaccharides of group A and C, whereas in the case of the B system the assay was more labile. The method is rapid and has a sensitivity comparable to that of radioimmunoassay. When studying paired sera from five recruits vaccinated with an A + C polysaccharide vaccine, significant rises in titers to both A and C polysaccharides were observed in all the postvaccination sera.

Immune lysis of reconstituted myelin basic protein--lipid vesicles and myelin vesicles.

Complement-mediated lysis of reconstituted lipid-myelin basic protein (BP) vesicles and myelin vesicles due to antibody raised against BP and isolated myelin is measured by determination of the amount of a water-soluble spin label, tempocholine chloride, released from the vesicles. The response is shown to be antigen-specific, antibody-dependent, and complement mediated. The relative response to different anti-BP antibody samples is similar to that determined by radioimmunoassay procedures. In contrast to immunoassays with BP in aqueous solution, this method measures immune recognition of the protein in either a synthetic or a natural membranous environment. This is important because this protein has been shown to have a different conformation when bound to lipid bilayers than in aqueous solution and its conformation depends on lipid composition. It is also a more rapid method because no separation of spin label still trapped in the vesicles and that released due to immune lysis is required. In synthetic membranes consisting of sphingomyelin, cholesterol, and an acidic lipid, either phosphatidylglycerol, phosphatidic acid, or phosphatidylserine, the response was greatest when the acidic lipid was phosphatidic acid. The response did not depend significantly on the antigen concentration expressed as molar ratio of BP to sphingomyelin, over the range 0.15:600 to 2:600, although it decreased at molar ratios less than 0.15:600. The antigen density required for immune lysis of vesicles containing this protein antigen is similar to that reported elsewhere for lipid antigens, although the time required for maximal lysis was greater. Both anti-BP and anti-myelin antibodies caused a greater specific complement-mediated response with synthetic vesicles than with myelin vesicles, which may be due to the different lipid and/or protein composition of myelin. Response was also obtained with the myelin vesicles, however, indicating that some determinants of BP can be recognized on the surface of the bilayer in isolated myelin by anti-BP.

Effect of streptolysin O and digitonin on egg lecithin/cholesterol vesicles

Artificial membrane vesicles (liposomes) have been used to study the lytic mechanism of the bacterial toxin, streptolysin O, compared to that of the well-known plant glycoside, digitonin. Two types of vesicle were prepared: large unilamellar vesicles and multilamellar liposomes. The vesicles were prepared with varying molar ratios of egg lecithin and cholesterol and loaded with the water-soluble spin label, TEMPO-choline chloride. Lysis of the vesicles was registered as release of spin label and monitored by change in the electron spin resonance (ESR) spectrum. In this system digitonin was able to lyse both large unilamellar vesicles and multilamellar liposomes. The effectiveness of lysis increased by increasing the percentage of cholesterol, but even at 0% cholesterol a significant level of lysis was observed by addition of a large enough concentration of digitonin. In contrast, no lysis was detected from multilamellar liposomes after exposure to streptolysin O, even when they consisted of 50 mol% cholesterol. On the other hand, large unilamellar vesicles could be lysed by streptolysin O, provided the cholesterol content was greater than 33%. At 67 mol% cholesterol in the membranes, the degree of lysis was diminished compared to 50%, which appeared to be optimal. This is the first demonstration of liposome lysis by streptolysin O and demonstrates the cholesterol specificity which has previously been shown by inhibition studies.

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 < −0.3 and log P > 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 labels. High fractions of lipid-soluble spin labels can be incorporated in unilamellar vesicles but the vesicle stability is diminished.

Changes in ram sperm membranes during epididymal transit

The sperm plasma membrane consists of three zones: a lipid bilayer that provides an impermeable barrier to uptake of extracellular, polar molecules; a charged lipid-water interface formed by the phospholipids and membrane-associated proteins; and a charged glycoprotein calyx with absorbed proteins. We quantitatively evaluated all three zones of membranes in testicular, cauda epididymal, and ejaculated ram sperm. Integrity of the lipid bilayer was assessed by measuring the aqueous isolated intracellular volume with water-soluble spin labels and electron spin resonance spectroscopy. Effects of rapid cooling to 0 °C (cold shock), of freezing on solid CO 2, and of butylated hydroxytoluene (BHT) on membrane integrity were evaluated. Testicular, cauda epididymal, and ejaculated sperm all had an isolated intracellular volume of 20–24 μm 3 per sperm. Isolated intracellular volume of testicular sperm was maintained (>90%) after cold shock, but reduced ~50% by freezing. BHT (1–3 m m) did not protect testicular sperm against membrane damage. Cauda epididymal and ejaculated sperm retained 20–30% of their isolated intracellular volume after cold shock but not freezing. However, 4 m m BHT prevented freeze damage of ejaculated but not cauda epididymal sperm. The lipid-water interface of the membrane was studied using surface-directed spin labels; the negative charge density was found to increase during epididymal transit. Net charge of the glycoprotein calyx was detected using whole-cell isoelectric focusing. Testicular, cauda epididymal, and ejaculated sperm isofocused at pH 5.2, 4.7, and 4.8 after insertion at pH 6 into the preformed pH gradient. Mixtures of testicular plus cauda epididymal or testicular plus ejaculated sperm were separable. We conclude that each of the three zones of the plasma membrane of sperm changes during epididymal maturation.

Monitoring the location profile of fluorophores in phosphatidylcholine bilayers by the use of paramagnetic quenching

Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short fange interaction we developed a spectroscopic method to characterize the molecular arrangement within the lipid membrane. Applied to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase. This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.

Antiliposome antisera activity against negatively charged phosphate amphiphils

Complement-immune lysis of liposomes loaded with water-soluble spin label 2,2,6,6-tetramethyl-piperidinoxy-choline chloride (TEMPO-choline) was used to measure specificity of rabbit antisera generated by complete adjuvant with liposomes consisting of sphingomyelin; cholesterol; dicetylphosphate; 5-N-thyroxine-2,4-dinitrophenyl -phosphatidylethanolamine (T 4-Dnp-PE) in the ratio 2.0:1.5:0.2:15. Antisera so generated induced complement lysis of the liposomes containing negatively charged amphiphils. No immune lysis of positively charged liposome was observed. This antiliposome specificity is retained in partially purified antibodies.

Paramagnetic fluorescence quenching in chlorophyll a containing vesicles: Evidence for the localization of chlorophyll

Spinprobes (fatty acids, androstane, cholestane) that differ in the location of the paramagnetic centre relative to the polar membrane surface have been incorporated into lipid vesicles containing chlorophyll a. Quenching of the Chl a fluorescence is observed following the Stern-Vollmer-relationship. Quenching is more effective with spinprobes carrying the nitroxide group near the polar head groups of the lipid moiety. Quenching is also observed using a water soluble spinlabel. The porphyrin ring of the Chl a molecules is suggested to be localized in the polar head group region of the bilayer membrane.

Immune lysis of spin label loaded liposomes incorporating cardiolipin; a new sensitive method for detecting anticardiolipin antibodies in syphilis serology

Liposomes prepared from a mixture of the pure lipids cholesterol, lecithin and cardiolipin (molar ratio 50/45/5), are able to bind antibodies directed against Treponema pallidum. When the liposomes are loaded with the water soluble spin label tempocholine chloride, the release of spin label from the liposomes can be monitored directly by observing changes in the paramagnetic resonance (ESR) spectrum from the spin label. The method offers a convenient technique for monitoring the complement-mediated lysis of liposomes, and may be applied in the serological diagnosis of syphilis, and a method for quantitative measurement of compliment.

Spin-label studies on the aqueous regions of phospholipid multilayers

Water-soluble spin labels were used to study dimyristoyllecithin (DML) phospholipid multilayers. Previous studies report that there is a "bound" water region associated with dimyristoyllecithin containing about 10 molecules of water per phospholipid, a "trapped" water region located between the lamellae containing approximately 11 molecules per phospholipid, and a "ftion show that certain water-soluble spin-label mol-cules have their motional properties differentially modified by these three water environements. Furthermore, the labels also reveal the onset of lipid-phase transitions even though they have high water solubility. A phosphate-containing spin label demonstrated strong an isotropic motion in the lipid-water system above the phase transition but not below. The addition of cholesterol to the DML-water system removed the anisotropic motion of 2,2,6,6-tetramehtyl-4-phosphopiperidine-N-oxyl (Tempophosphate) and obscured the detection bound, trapped, and free water. In addition to the change-charge interactions between Tempophosphate and DML, two other spin labels were used both in the charged and uncharged states. 2,2,6,6-Tetramethyl-4-aminopiperidine-N-oxyl (Tempamine) in the charged state showed extremely strong anisotropic motion, presumably due to the interaction between the charged amine and the phosphate group of DML. When only partially charged, Tempamine showed much less anisotropic motion. PCA was analyzed at pH values where the carboxyl group was protonated and unprotonated. The resulting interaction was different at the two pH values. These water-soluble spin labels mimic ionic or nonionic solutes. Upon freezing, the spin labels are shown to be expelled from the ice regions into the remaining aqueous regions. The usefulness of this approach in studying solute behavior when freezing occurs and potential studies involving aqueous regions of cytoplasm are considered.

Changes in the restriction of molecular rotational diffusion of water-soluble spin labels during fatty acid starvation of yeast

Yeast mutants lacking fatty acid synthetase activity (fas-) die when deprived of saturated fatty acid under conditions which are otherwise growth-supporting. The spin label technique is used to show that restriction of molecular rotational diffusion of spin label molecules dissolved in aqueous zones increases several fold under conditions of fatty acid starvation while the apparent physical state of cellular hydrocarbon zones remains essentially unchanged. We focus attention on the cellular aqueous interior as the potential site of alteration under selective starvation conditions. Correspondences exist between restriction of molecular motion of water soluble spin labels dissolved in the cell and loss of cell viability. The correspondences to changes in the molecular motion of hydrocarbon soluble spin labels are much less or are not detectable.

A spin-label study of the viscosity profile of sarcoplasmic reticular vesicles

Sarcoplasmic reticular vesicles were prepared from both lobster and rabbit muscle. A variety of water-soluble, lipid-soluble, and alkylating spin labels were used to treat the sarcoplasmic reticular vesicles. All spin label analyses were carried out with and without NiCl 2. Nickel is used to remove spin label signal originating from outside of, on the surface of, or localized in the outermost part of the outer bilayer half of the sarcoplasmic reticular membrane. We conclude that the hydrocarbon portion of sarcoplasmic reticular vesicles has symmetry in regard to the physical properties that limit spin label motion; however, we find that the membrane interface limits spin label motion more on the inner surface than the outer surface and that the trapped aqueous volume which is sampled by water soluble spin labels inside the vesicle enclosure limits spin label motion much more than the average aqueous medium outside.