Electron Paramagnetic Resonance Spin-labeling Methods Research Articles

Association of Alpha-Crystallin with Human Cortical and Nuclear Lens Lipid Membrane Increases with the Grade of Cortical and Nuclear Cataract.

Eye lens α-crystallin has been shown to become increasingly membrane-bound with age and cataract formation; however, to our knowledge, no studies have investigated the membrane interactions of α-crystallin throughout the development of cataracts in separated cortical membrane (CM) and nuclear membrane (NM) from single human lenses. In this study, four pairs of human lenses from age-matched male and female donors and one pair of male lenses ranging in age from 64 to 73 years old (yo) were obtained to investigate the interactions of α-crystallin with the NM and CM throughout the progression of cortical cataract (CC) and nuclear cataract (NC) using the electron paramagnetic resonance spin-labeling method. Donor health history information (diabetes, smoker, hypertension, radiation treatment), sex, and race were included in the data analysis. The right eye lenses CM and NM investigated were 64 yo male (CC: 0), 68 yo male (CC: 3, NC: 2), 73 yo male (CC: 1, NC: 2), 68 yo female (CC: 3, NC: 2), and 73 yo female (CC: 1, NC: 3). Similarly, left eye lenses CM and NM investigated were 64 yo male (CC: 0), 68 yo male (CC: 3, NC: 2), 73 yo male (CC: 2, NC: 3), 68 yo female (CC: 3, NC: 2), and 73 yo female (CC: 1, NC: 3). Analysis of α-crystallin binding to male and female eye lens CM and NM revealed that the percentage of membrane surface occupied (MSO) by α-crystallin increases with increasing grade of CC and NC. The binding of α-crystallin resulted in decreased mobility, increased order, and increased hydrophobicity on the membrane surface in male and female eye lens CM and NM. CM mobility decreased with an increase in cataracts for both males and females, whereas the male lens NM mobility showed no significant change, while female lens NM showed increased mobility with an increase in cataract grade. Our data shows that a 68 yo female donor (long-term smoker, pre-diabetic, and hypertension; grade 3 CC) showed the largest MSO by α-crystallin in CM from both the left and right lens and had the most pronounced mobility changes relative to all other analyzed samples. The variation in cholesterol (Chol) content, size and amount of cholesterol bilayer domains (CBDs), and lipid composition in the CM and NM with age and cataract might result in a variation of membrane surface mobility, membrane surface hydrophobicity, and the interactions of α-crystallin at the surface of each CM and NM. These findings provide insight into the effect of decreased Chol content and the reduced size and amount of CBDs in the cataractous CM and NM with an increased binding of α-crystallin with increased CC and NC grade, which suggests that Chol and CBDs might be a key component in maintaining lens transparency.

Phospholipid acyl chain length and degree of unsaturation modulate α-crystallin binding to the membrane

The binding of α-crystallin to the eye lens’s fiber cell plasma membrane increases with age and cataract. It has also been reported that the primary binding site of α-crystallin in the lens’s membrane is phospholipids (PLs). However, it is unclear whether PL acyl chain length and degree of unsaturation influence α-crystallin binding. We have used electron paramagnetic resonance spin-labeling methods to investigate the binding of α-crystallin with cholesterol (Chol)/phosphatidylcholine (PC) membrane of different chain lengths and degree of unsaturation (i.e., 14:0 PC, 18:0 18:1 PC, 18:1 PC, and 16:0 20:4 PC) and in the presence and absence of 23 mol% Chol.

Cholesterol and cholesterol bilayer domains inhibit binding of alpha-crystallin to the membranes made of the major phospholipids of eye lens fiber cell plasma membranes

The concentration of α-crystallin decreases in the eye lens cytoplasm, with a corresponding increase in membrane-bound α-crystallin during cataract formation. The eye lens's fiber cell plasma membrane consists of extremely high cholesterol (Chol) content, forming cholesterol bilayer domains (CBDs) within the membrane. The role of high Chol content in the lens membrane is unclear. Here, we applied the continuous-wave electron paramagnetic resonance spin-labeling method to probe the role of Chol and CBDs on α-crystallin binding to membranes made of four major phospholipids (PLs) of the eye lens, i.e., phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Small unilamellar vesicles (SUVs) of PC, SM*, and PS with 0, 23, 33, 50, and 60 mol% Chol and PE* with 0, 9, and 33 mol% Chol were prepared using the rapid solvent exchange method followed by probe-tip sonication. The 1 mol% CSL spin-labels used during SUVs preparation distribute uniformly within the Chol/PL membrane, enabling the investigation of Chol and CBDs' role on α-crystallin binding to the membrane. For PC, SM*, and PS membranes, the binding affinity (Ka) and the maximum percentage of membrane surface occupied (MMSO) by α-crystallin decreased with an increase in Chol concentration. The Ka and MMSO became zero at 50 mol% Chol for PC and 60 mol% Chol for SM* membranes, representing that complete inhibition of α-crystallin binding was possible before the formation of CBDs within the PC membrane but only after the formation of CBDs within the SM* membrane. The Ka and MMSO did not reach zero even at 60 mol% Chol in the PS membrane, representing CBDs at this Chol concentration were not sufficient for complete inhibition of α-crystallin binding to the PS membrane. Both the Ka and MMSO were zero at 0, 9, and 33 mol% Chol in the PE* membrane, representing no binding of α-crystallin to the PE* membrane with and without Chol. The mobility parameter profiles decreased with an increase in α-crystallin binding to the membranes; however, the decrease was more pronounced for the membrane with lower Chol concentration. These results imply that the membranes become more immobilized near the headgroup regions with an increase in α-crystallin binding; however, the Chol antagonizes the capacity of α-crystallin to decrease the mobility near the headgroup regions of the membranes. The maximum splitting profiles remained the same with an increase in α-crystallin concentration, but there was an increase in the maximum splitting with an increase in the Chol concentration in the membranes. It implies that membrane order near the headgroup regions does not change with an increase in α-crystallin concentration but increases with an increase in Chol concentration in the membrane. Based on our data, we hypothesize that the Chol and CBDs decrease hydrophobicity (increase polarity) near the membrane surface, inhibiting the hydrophobic binding of α-crystallin to the membranes. Thus, our data suggest that Chol and CBDs play a positive physiological role by preventing α-crystallin binding to lens membranes and possibly protecting against cataract formation and progression.

Detection of cholesterol bilayer domains in intact biological membranes: Methodology development and its application to studies of eye lens fiber cell plasma membranes

Four purported lipid domains are expected in plasma membranes of the eye lens fiber cells. Three of these domains, namely, bulk, boundary, and trapped lipids, have been detected. The cholesterol bilayer domain (CBD), which has been detected in lens lipid membranes prepared from the total lipids extracted from fiber cell plasma membranes, has not yet been detected in intact fiber cell plasma membranes. Here, a saturation-recovery electron paramagnetic resonance spin-labeling method has been developed that allows identification of CBDs in intact fiber cell plasma membranes of eye lenses. This method is based on saturation-recovery signal measurements of the cholesterol-analog spin label located in the lipid bilayer portion of intact fiber cell membranes as a function of the partial pressure of molecular oxygen with which the samples are equilibrated. The capabilities and limitations of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses where CBDs were detected in porcine nuclear intact membranes for which CBDs were also detected in lens lipid membranes. CBDs were not detected in porcine cortical intact and lens lipid membranes. CBDs were detected in intact membranes isolated from both cortical and nuclear fiber cells of lenses obtained from human donors. The cholesterol content in fiber cell membranes of these donors was always high enough to induce the formation of CBDs in cortical as well as nuclear lens lipid membranes. The results obtained for intact membranes, when combined with those obtained for lens lipid membranes, advance our understanding of the role of high cholesterol content and CBDs in lens biology, aging, and/or cataract formation.

Is the cholesterol bilayer domain a barrier to oxygen transport into the eye lens?

In the eye lens, the oxygen partial pressure is very low and the cholesterol (Chol) content in cell membranes is very high. Disturbance of these quantities results in cataract development. In human lens membranes, both bulk phospholipid-Chol domains and the pure Chol bilayer domains (CBDs) were experimentally detected. It is hypothesized that the CBD constitutes a significant barrier to oxygen transport into the lens. Transmembrane profiles of the oxygen diffusion-concentration product, obtained with electron paramagnetic resonance spin-labeling methods, allow evaluation of the oxygen permeability (PM) of phospholipid membranes but not the CBD. Molecular dynamics simulation can independently provide components of the product across any bilayer domain, thus allowing evaluation of the PM across the CBD. Therefore, to test the hypothesis, MD simulation was used. Three bilayers containing palmitoyl-oleoyl-phosphorylcholine (POPC) and Chol were built. The pure Chol bilayer modeled the CBD, the 1:1 POPC-Chol bilayer modeled the bulk membrane in which the CBD is embedded, and the POPC bilayer was a reference. To each model, 200 oxygen molecules were added. After equilibration, the oxygen concentration and diffusion profiles were calculated for each model and multiplied by each other. From the respective product profiles, the PM of each bilayer was calculated. Favorable comparison with experimental data available only for the POPC and POPC-Chol bilayers validated these bilayer models and allowed the conclusion that oxygen permeation across the CBD is ~10 smaller than across the bulk membrane, supporting the hypothesis that the CBD is a barrier to oxygen transport into the eye lens.

Saturation recovery EPR spin-labeling method for quantification of lipids in biological membrane domains.

The presence of integral membrane proteins induces the formation of distinct domains in the lipid bilayer portion of biological membranes. Qualitative application of both continuous wave (CW) and saturation recovery (SR) electron paramagnetic resonance (EPR) spin-labeling methods allowed discrimination of the bulk, boundary, and trapped lipid domains. A recently developed method, which is based on the CW EPR spectra of phospholipid (PL) and cholesterol (Chol) analog spin labels, allows evaluation of the relative amount of PLs (% of total PLs) in the boundary plus trapped lipid domain and the relative amount of Chol (% of total Chol) in the trapped lipid domain [M. Raguz, L. Mainali, W. J. O'Brien, and W. K. Subczynski (2015), Exp. Eye Res., 140:179-186]. Here, a new method is presented that, based on SR EPR spin-labeling, allows quantitative evaluation of the relative amounts of PLs and Chol in the trapped lipid domain of intact membranes. This new method complements the existing one, allowing acquisition of more detailed information about the distribution of lipids between domains in intact membranes. The methodological transition of the SR EPR spin-labeling approach from qualitative to quantitative is demonstrated. The abilities of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses. Statistical analysis (Student's t-test) of the data allowed determination of the separations of mean values above which differences can be treated as statistically significant (P ≤ 0.05) and can be attributed to sources other than preparation/technique.

Lipid-Protein Interactions in Fiber Cell Plasma Membrane Isolated From Human and Porcine Eye Lenses

The protein content in human lens fiber cell plasma membranes is extremely high, increases with age, and is higher in the nucleus than in cortex. This strongly affects the organization and physical properties of the lipid bilayer portion of intact fiber cell plasma membranes including the alkyl chain order, fluidity, hydrophobicity, and oxygen transport parameter. Investigated membranes were isolated from the eye lenses of pigs and human donors. They were studied using electron paramagnetic resonance spin-labeling method that provides an opportunity to discriminate coexisting lipid domains and to evaluate the relative amounts of phospholipids and cholesterol in them. Fiber-cell membranes were found to contain three distinct lipid environments: bulk, boundary and trapped domains that appear due to the presence of membrane proteins. In nuclear membranes the amount of boundary and trapped phospholipids as well as the amount of cholesterol in trapped lipid domains was greater than that in cortical membranes. Cholesterol was to a large degree excluded from trapped lipid domains in cortical membranes. The difference between the amounts of lipids in domains uniquely formed due to the presence of membrane proteins increased with the donors‘ age. The rigidity of nuclear membranes was greater than that of cortical membranes for all age groups. The nuclear fiber cell plasma membranes were less permeable to oxygen than cortical membranes and both of them become less permeable to oxygen with age. In nuclear porcine membranes the amounts of lipids in domains created due to the presence of membrane proteins were greater than those in cortical membranes and the differences were larger than the differences observed for human membranes. Lipids in porcine nuclear fiber cell membranes were more rigid and less permeable to oxygen than in human nuclear membranes.

Saturation-Recovery EPR Spin-Labeling Method for Quantification of Lipids in Domains of Biological Membranes

The presence of integral membrane proteins in biological membranes induces the formation of distinct domains in the lipid bilayer portion of these membranes. We were able to discriminate the existence of three lipid environments in intact fiber cell plasma membranes of the eye lens, namely bulk, boundary, and trapped lipids. However, our qualitative approach did not allow quantification of lipids in each domain. Recently, based on the continuous wave (CW) electron paramagnetic resonance (EPR) spectra of phospholipid analog and cholesterol analog spin labels, we developed a method that allows evaluation of the relative amount of phospholipids and cholesterol in the bulk lipid domain and in the boundary plus trapped lipid domain [M. Raguz, L. Mainali,W. J. O’Brien and W. K. Subczynski, (2015), Exp. Eye Res., 140:179-186]. Here, we will present a new approach that, based on saturation-recovery (SR) EPR, allows evaluation of the relative amount of phospholipids and cholesterol in the bulk plus boundary lipid domain and in the trapped lipid domain. Spectrometer conditions for the successful application of this method to quantify lipids in membrane domains are described. These two methods clearly demonstrate that the method's time window allows the separation of results from the different domains. CW EPR mixes results from the boundary and trapped lipids, while SR EPR mixes results from the bulk and boundary lipids. These two methods complement each other, providing a more complete picture of lipid lateral organization in intact membranes. The abilities of these methods are illustrated in the intact fiber cell plasma membranes from porcine eye lenses.Acknowledgments: This work was supported by grants EY015526, EB001980, and EY001931 from the National Institutes of Health.

Amounts of phospholipids and cholesterol in lipid domains formed in intact lens membranes: Methodology development and its application to studies of porcine lens membranes

An electron paramagnetic resonance spin-labeling method has been developed that allows quantitative evaluation of the amounts of phospholipids and cholesterol in lipid domains of intact fiber-cell plasma membranes isolated from cortical and nuclear regions of eye lenses. The long term goal of this research is the assessment of organizational changes in human lens fiber cell membranes that occur with age and during cataract development. The measurements needed to be performed on lens membranes prepared from eyes of single donors and from single eyes. For these types of studies it is necessary to separate the age/cataract related changes from preparation/technique related changes. Human lenses differ not only because of age, but also because of the varying health histories of the donors. To solve these problems the sample-to-sample preparation/technique related changes were evaluated for cortical and nuclear lens membranes prepared from single porcine eyes. It was assumed that the differences due to the age (animals were two year old) and environmental conditions for raising these animals were minimal. Mean values and standard deviations from preparation/technique changes for measured amounts of lipids in membrane domains were calculated. Statistical analysis (Student's t-test) of the data also allowed determining the differences of mean values which were statistically significant with P ≤ 0.05. These differences defined for porcine lenses will be used for comparison of amounts of lipids in domains in human lens membranes prepared from eyes of single donors and from single eyes. Greater separations will indicate that differences were statistically significant with (P ≤ 0.05) and that they came from different than preparation/technique sources. Results confirmed that in nuclear porcine membranes the amounts of lipids in domains created due to the presence of membrane proteins were greater than those in cortical membranes and the differences were larger than the differences observed for human intact fiber cell membranes [Raguz, M. Mainali, L., O'Brien, W.J., and Subczynski, W.K. (2015) Exp. Eye Res.]. Lipids in porcine nuclear fiber cell plasma membranes were more rigid and less permeable to oxygen than in human nuclear membranes. Most likely the significant differences in the lipid composition were responsible for the observed differences.

Lipid domains in intact fiber-cell plasma membranes isolated from cortical and nuclear regions of human eye lenses of donors from different age groups

The results reported here clearly document changes in the properties and the organization of fiber-cell membrane lipids that occur with age, based on electron paramagnetic resonance (EPR) analysis of lens membranes of clear lenses from donors of age groups from 0 to 20, 21 to 40, and 61 to 80 years. The physical properties, including profiles of the alkyl chain order, fluidity, hydrophobicity, and oxygen transport parameter, were investigated using EPR spin-labeling methods, which also provide an opportunity to discriminate coexisting lipid domains and to evaluate the relative amounts of lipids in these domains. Fiber-cell membranes were found to contain three distinct lipid environments: bulk lipid domain, which appears minimally affected by membrane proteins, and two domains that appear due to the presence of membrane proteins, namely boundary and trapped lipid domains. In nuclear membranes the amount of boundary and trapped phospholipids as well as the amount of cholesterol in trapped lipid domains increased with the donors' age and was greater than that in cortical membranes. The difference between the amounts of lipids in domains uniquely formed due to the presence of membrane proteins in nuclear and cortical membranes increased with the donors' age. It was also shown that cholesterol was to a large degree excluded from trapped lipid domains in cortical membranes. It is evident that the rigidity of nuclear membranes was greater than that of cortical membranes for all age groups. The amount of lipids in domains of low oxygen permeability, mainly in trapped lipid domains, were greater in nuclear than cortical membranes and increased with the age of donors. These results indicate that the nuclear fiber cell plasma membranes were less permeable to oxygen than cortical membranes and become less permeable to oxygen with age. In clear lenses, age-related changes in the lens lipid and protein composition and organization appear to occur in ways that increase fiber cell plasma membrane resistance to oxygen permeation.

Cholesterol Bilayer Domain in Phospholipid Bilayer Membranes can be Detected by Confocal Microscope

The unique feature of the eye lens fiber-cell plasma membrane is its extremely high cholesterol content; cholesterol/phospholipid molar ratio can be as high as 4 in human lens nucleus. Cholesterol saturates bulk phospholipid bilayers and induces formation of immiscible cholesterol bilayer domains (CBDs) within membranes. The presence of CBDs plays crucial role ensuring that surrounding phospholipid bilayers are saturated with cholesterol which keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, CBDs help to maintain lens-membrane homeostasis when the membrane phospholipid composition changes significantly with age. Our previous experiments were based on electron paramagnetic resonance spin-labeling methods. They allowed to detect CBDs in model and lens lipid membranes at high cholesterol content (1,2). In current experiments the giant unilamellar vesicles made of cholesterol/distearoylphosphatidylcholine (Chol/DSPC) mixtures, with mixing ration from 0.5 to 5 and labeled with phospholipid analog (1,1′-Dioctadecyl-3,3,3’,3′-Tetramethylindocarbocyanine-5,5′-Disulfonic Acid) and cholesterol analog (23-(dipyrrometheneboron difluoride)-24-norcholesterol) fluorescent probes, were prepared using the electroformation method (3). Confocal microscopy experiment allowed us to confirm that membranes with higher Chol/DSPC molar ratio contain two distinct lipid environments, the bulk phospholipid-cholesterol domain (PCD) and the CBD. The amount of CBD was greater in membranes with higher Chol/DSPC ratio. According to these results, CBD is always located on the top of the giant vesicle. Rationale for this discovery is that CBD has smaller mass per unit area of the vesicle compared with PCD. Additionally, it can be concluded that vesicles with higher Chol/DSPC molar ratio are more rigid and show less wobbling motion in water environment.1. Raguz et al. CPL 2011 164(8):819-29.2. Mainali et al. BBA 2013 1828(6):1432-40.3. Veatch SL. Methods Mol Biol. 2007 398:59-72.AcknowledgementsSupported by NIH grants EY015526 and EY001931.

Properties of membranes derived from the total lipids extracted from clear and cataractous lenses of 61-70-year-old human donors.

Human lens-lipid membranes prepared from the total lipids extracted from clear and cataractous lens cortexes and nuclei of 61-70-year-old donors by use of a rapid solvent-exchange method were investigated. The measured cholesterol-to-phospholipid (Chol/PL) molar ratio in these membranes was 1.8 and 4.4 for cortex and nucleus of clear lenses, respectively, and 1.14 and 1.45 for cataractous lenses. Properties and organization of the lipid bilayer were investigated by use of electron paramagnetic resonance spin-labeling methods. Formation of Chol crystals was confirmed by use of differential scanning calorimetry. Pure cholesterol bilayer domains (CBDs) were formed in all the membranes investigated. It was shown that in clear lens membranes of the nucleus, Chol exists in three different environments: (1) dispersed in phospholipid bilayers (PCDs), (2) in CBDs, and (3) in Chol crystals. In clear lens membranes of the cortex, and in cortical and nuclear cataractous lens membranes, Chol crystals were not detected, because of the lower Chol content. Profiles of membrane properties (alkyl-chain order, fluidity, oxygen transport, and hydrophobicity) across the PCD were very similar for clear and cataractous membranes. Profiles ofthe oxygen transportparameter across the CBD were, however, different for cortical clear and cataractous membranes-the amount and size of CBDs was less in cataractous membranes. These results suggest that high Chol content, formation of CBDs, and formation of Chol crystals should not be regarded as major predispositions for the development of age-related cataracts.

Lipid–protein interactions in plasma membranes of fiber cells isolated from the human eye lens

The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O’Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed.

Phospholipid-Cholesterol Bilayers, Cholesterol Bilayer Domains, and Cholesterol Crystals were Detected in Lipid Dispersion Prepared from Lipids Extracted from Lens Nucleus of Old Human Donors

Human lens lipid membranes prepared using a rapid solvent exchange method from the total lipids extracted from the clear lens cortex and nucleus of 61- to 70-year-old donors were investigated. The measured cholesterol-to-phospholipid (Chol/PL) molar ratio in these preparations was extremely high, showing values of 1.8 and 4.4 for cortex and nucleus, respectively. We expect that at this elevated Chol content, the entire membrane will become saturated with Chol (as in the case of the cortical membrane) and the excess of Chol will form Chol crystals, presumably outside the membrane (as in the case of the nuclear membrane). Properties and organization of the lipid bilayer were investigated using electron paramagnetic resonance spin-labeling methods. Formation of Chol crystals was confirmed using the differential scanning calorimetry. We showed that in the lipid dispersion prepared from nuclear lipids Chol exists in three distinguished environments: (1) Chol dispersed in PL bilayer, (2) Chol in non-crystalline membrane domains (cholesterol bilayer domains, CBDs), and (3) Chol in crystals. In cortical membranes, because of the lower Chol content, Chol crystals were not detected. Amounts of Chol in CBDs were almost the same in cortical and nuclear membranes which indicates that Chol content in both membranes is close or exceeds the Chol solubility thresholds in these membranes. Profiles of cortical and nuclear membrane properties (alkyl-chain order, fluidity, oxygen transport parameter, and hydrophobicity) were very similar to each other and to those reported for cortical and nuclear lens lipid membranes of 41- to 60-year-old donors reported earlier. This confirms our earlier statement that saturation with Chol determines properties of the PL bilayer with the minor effect of the PL composition.

Use of electron paramagnetic resonance to solve biochemical problems.

Electron paramagnetic resonance (EPR) spectroscopy is a very powerful biophysical tool that can provide valuable structural and dynamic information about a wide variety of biological systems. The intent of this review is to provide a general overview for biochemists and biological researchers of the most commonly used EPR methods and how these techniques can be used to answer important biological questions. The topics discussed could easily fill one or more textbooks; thus, we present a brief background on several important biological EPR techniques and an overview of several interesting studies that have successfully used EPR to solve pertinent biological problems. The review consists of the following sections: an introduction to EPR techniques, spin-labeling methods, and studies of naturally occurring organic radicals and EPR active transition metal systems that are presented as a series of case studies in which EPR spectroscopy has been used to greatly further our understanding of several important biological systems.

Phase-Separation and Domain-Formation in Cholesterol-Sphingomyelin Mixture: Pulse-EPR Oxygen Probing

Membranes made of Chol/ESM (cholesterol/egg sphingomyelin) mixtures were investigated using saturation-recovery electron paramagnetic resonance spin-labeling methods, in which bimolecular collisions of relaxation agents (oxygen or nickel ethylenediamine diacetic acid) with spin labels are measured. Liquid-disordered (ld) and liquid-ordered (lo) phases, and cholesterol bilayer domains (CBDs) were discriminated and characterized by profiles of the oxygen transport parameter (OTP). In the ld phase, coexisting with the lo phase, the OTP profile is bell-shaped and lies above that in the pure ESM membrane. Changes in the OTP profile across the lo phase are complex. When the lo phase coexists with the ld phase, the OTP profile is similar to that across the pure ESM membrane but with a steeper bell shape. With an increase in cholesterol concentration (up to the cholesterol-solubility threshold), the profile becomes rectangular, with low OTP values from the membrane surface to the depth of C9, and high values in the membrane center. This approximately threefold increase in the OTP occurs at the depth at which the rigid ring structure of cholesterol is immersed. Further addition of cholesterol and the formation of the CBD does not affect the OTP profile across the lo phase. OTP values in the CBD are significantly lower than in the lo phase.

Carotenoid-membrane interactions in liposomes: effect of dipolar, monopolar, and nonpolar carotenoids.

Electron paramagnetic resonance (EPR) spin-labeling methods were used to study the effects of carotenoids on the physical properties of saturated phosphatidylcholine (PC) membranes to evaluate the contribution of the terminal hydroxyl groups of xanthophyll molecules to the carotenoid-membrane interaction. Effects of the dipolar, terminally dihydroxylated carotenoid lutein on membrane phase transition, fluidity, order, and polarity were compared with those of monopolar (beta-cryptoxanthin) and nonpolar (beta-carotene) carotenoids. These effects were monitored at the membrane center as a function of the amount of the carotenoid added to the sample and as a function of temperature for fluid-phase membranes. PC membranes with different thickness (from 12 to 22 carbons in alkyl chains) were used. Carotenoids shifted to lower temperatures and broadened the main phase transition of PC membranes. They decreased the membrane fluidity and increased the order of alkyl chains. Carotenoids also increased the hydrophobicity of the membrane interior. These effects were the strongest for lutein, significantly weaker for beta-cryptoxanthin, and negligible for beta-carotene. They decreased with the increase of the membrane thickness. Presented results suggest that anchoring of carotenoid molecules at the opposite membrane surfaces by polar hydroxyl groups is significant in enhancing their effects on membrane properties. This manuscript also shows the ability of EPR spin-labeling methods to monitor different membrane properties that can be applied in biotechnological studies with the use of liposomes.

Erythrocyte membrane fluidity changes in psoriasis: an EPR study

The aim of the study was to find the cause of membrane fluidity decrease in psoriasis, observed by other authors, in different types of cells and its clinical consequences. To this end, we have examined the influence of different clinical and biochemical factors on erythrocyte membrane fluidity changes in psoriatic patients. Membrane fluidity was studied by the electron paramagnetic resonance spin-labeling method. The data revealed that the decrease of membrane fluidity corresponded with exacerbation of skin lesions. The results clearly indicate that the increased lipid peroxidation may be the essential mechanism of membrane fluidity decrease in psoriasis.

Physical properties of lipid bilayer membranes: relevance to membrane biological functions.

Over the last 25 years one of us (WKS) has been investigating physical properties of lipid bilayer membranes. In 1991 a group led by WKS was organized into the Laboratory of Structure and Dynamics of Biological Membranes, the effective member of which is AW. Using mainly the electron paramagnetic resonance (EPR) spin-labeling method, we obtained unexpected results, which are significant for the better understanding of the functioning of biological membranes. We have developed a new pulse EPR spin-labeling method for the detection of membrane domains and evaluation of lipid exchange rates. This review will be focused on our main results which can be summarized as follows: (1) Unsaturation of alkyl chains greatly reduces the ordering and rigidifying effects of cholesterol although the unsaturation alone gives only minor fluidizing effects, as observed by order and reorientational motion, and rather significant rigidifying effects, as observed by translational motion of probe molecules; (2) Fluid-phase model membranes and cell plasma membranes are not barriers to oxygen and nitric oxide transport; (3) Polar carotenoids can regulate membrane fluidity in a way similar to cholesterol; (4) Formation of effective hydrophobic barriers to the permeation of small polar molecules across membranes requires alkyl chain unsaturation and/or the presence of cholesterol; (5) Fluid-phase micro-immiscibility takes place in cis-unsaturated phosphatidylcholine-cholesterol membranes and induces the formation of cholesterol-rich domains; (6) In membranes containing high concentrations of transmembrane proteins a new lipid domain is formed, with lipids trapped within aggregates of proteins, in which the lipid dynamics is diminished to the level of gel-phase.

Influence of surfactant mixtures on intercellular lipid fluidity and skin barrier function

Background/aims: Surfactant mixtures are used in cosmetic and pharmaceutical formulas in order to establish product efficacy while maintaining mildness and skin lipids. The electron paramagnetic resonance (EPR) technique of the spin labeling method with a nitroxide spin probe is a valuable method in the study of biological membranes. The objective of this study was to define the influence of surfactant mixtures on intercellular lipid fluidity and correlate EPR spectral data with in vivo safety data.Methods: EPR experiment: EPR spectra of 5‐doxyl stearic acid (5‐DSA) labeled stratum corneum treated with sodium lauryl sulfate (SLS), sodium lauroyl glutamate (SLG) and their mixtures were measured and order parameters were calculated. Clinical testing: Fifteen healthy volunteers free of skin disease and with no history of atopic dermatitis were treated with SLS solutions (0.25%, 0.50%, 0.75%, 1.00%), 1.00% SLG solution and 1.00% surfactant mixture solutions: 0.75% SLS+0.25% SLG, 0.50% SLS+0.50% SLG, 0.25% SLS+0.75% SLG. One hundred μl of solution was applied using a polypropylene chamber for 24 h. Transepidermal water loss (TEWL) was measured with an evaporimeter before and after the application of surfactant solutions and each site was also visually graded according to Lee (1).Results: The order parameter (S) calculated from 1.00 %wt SLS treated stratum corneum was 0.56 ± 0.03, indicating disordering of lipid structure. On the contrary, the high S value (0.82 ± 0.02) for 1.00 %wt SLG suggests a reduced effect on the structured lipid, almost equaling the value of water. Treatment with 0.25 %wt, 0.50 %wt and 0.75 %wt SLS solutions revealed intermediate levels between 1.00 %wt SLG and SLS. The order parameters at each SLS concentration (0.25, 0.50, 0.75 and 1.00 %wt SLS) with 1.00 %wt SLG showed higher values than those of SLS only solutions. There were statistically significant differences between with and without 1.00 %wt SLG (P < 0.05). These results suggest that the addition of 1.00 %wt SLG inhibits the fluidization of intercellular lipid induced by SLS. The visual scores and TEWL values of 1.00% SLG solution were lower than those of the other test solutions (except for the vehicle control: deionized water). The 1.00% surfactant mixture solutions showed lower visual scores and TEWL values of the 1.00% SLS solution. An increase of SLG concentration decreased the visual scores and TEWL values. Order parameter S obtained from EPR spectra correlated with the clinical study. The correlation coefficient (r2) of visual score and TEWL values was 0.73 and 0.83, respectively.Conclusion: SLS disorder (fluidity) intercellular lipids at low concentrations, such as 0.25 %wt, presumably due to the SLS molecules being intercalated into the intercellular lipids. However, EPR spectral data suggest that the addition of 1.00 %wt SLG to an SLS solution (<1.00 %wt) inhibits the fluidization of intercellular lipid induced by SLS. A reasonable correlation between order parameters and human clinical data (visual scores and TEWL values) was observed (r2=0.73 and 0.83, respectively). The use of the EPR spin labeling method for predicting the fluidity of stratum corneum should give further insight into the mechanism of epidermal barrier disruption by surfactants and possibly other chemicals.