Spin-label saturation-recovery EPR at W-band: Applications to eye lens lipid membranes
Graphical abstract
Highlights
► Fluidity and oxygen transport parameter profiles for lens membranes at W-band. ► Discrimination and characterization of membrane domains at W-band. ► Feasibility for performing experiments on eye lenses from a single human donor.
Introduction
The saturation-recovery (SR) EPR method was pioneered at the National Biomedical EPR Center in Milwaukee [1]. The X-band (9.4 GHz) SR spectrometer, which is equipped with a loop-gap resonator (LGR), has been significantly improved in recent years [2], [3]. Other EPR spectrometers built in the EPR Center allow SR measurements at microwave frequencies from 2 to 94 GHz [3], [4]. In previous papers [3], [4], [5], we showed that (1) the T1 values of water-soluble spin labels as well as lipid-type spin labels in membranes depend on microwave frequency (being longest at Q-band (35 GHz)), and (2) that the effect of collisions between oxygen and spin-labels on the measured T1 values are independent of frequency at all microwave frequencies.
Recently, we used EPR spin-labeling methods, including the SR approach, to study organization and dynamics of lens lipid membranes from different species (6-month-old calf and pig [6], [7], [8]), from animals of different ages (6-month-old and 2-year-old cow [6], [7], [9]), and from different eye regions (cortex and nucleus of a 2-year-old cow [9]). These membranes are overloaded with cholesterol, which not only saturates phospholipid bilayers but also leads to the formation of cholesterol bilayer domains (CBDs) within the membrane [8], [9]. EPR spin-labeling methods provide a unique opportunity for determining the lateral organization of lens lipid membranes including coexisting membrane domains [10], [11]. They also provide a number of unique approaches for determining several important membrane properties as a function of bilayer depth including alkyl chain order [12], hydrophobicity [13], and oxygen diffusion–concentration product (called the oxygen transport parameter) [14]. In some cases, these properties can be obtained in coexisting membrane domains without the need for physical separation [10], [11]. EPR spin-labeling methods also make it possible to obtain molecular-level information on the organization and dynamics of cholesterol molecules in the CBD as well as information on physical properties of this domain [15]. This type of information cannot be obtained by differential scanning calorimetry (DSC) [16], [17], [18], X-ray, or neutron diffraction [16], [17], [19], [20], [21] methods, which also have been applied to investigate the lateral organization of lens lipid membranes and intact lens membranes.
All previous investigations were carried out at X-band using conventional and SR EPR spectrometers with an LGR that has a sample volume of 3 μL. To complete all measurements and obtain detailed profiles, lipids were extracted from 50 to 100 eye lenses. It is not difficult to obtain these numbers of similar eye lenses (age is the major criterion) from a meat-packing plant. Human lenses however are more precious and more difficult to obtain in these numbers from eye banks. A more serious problem is that human lenses can be different not only because of age, but also because of varying health history of the donor. The best solution of this problem will be to perform all measurements on samples prepared from one or two eyes from a single donor.
Here, we present results that demonstrate the feasibility of such measurements. Profiles of lens lipid membrane properties that were obtained using spin-label EPR at X-band with an LGR with a sample volume of 3 μL can also be obtained at W-band with the LGR with a sample volume of 30 nL. Thus, the total amount of sample can be 100 times smaller at W-band than at X-band. Results at W-band and X-band include profiles of the membrane fluidity and oxygen transport parameter, as well as data on discrimination of coexisting membrane domains. Additionally, results are reported about properties of 2-year-old porcine cortical and nuclear membranes, which complement the published data describing properties of 2-year-old bovine cortical and nuclear membranes [9], increasing our knowledge about organization and dynamics of lens lipid membranes from different species.
In these studies, phospholipid- and cholesterol-analog spin labels (see Fig. 2 in Ref. [8] for structures and approximate localization in the lipid bilayer) are incorporated in the membrane with the nitroxide moiety, which gives rise to the observed EPR signal at specific depths and in specific membrane domains. These spin labels have molecular structures that are similar to parent phospholipids or cholesterol and therefore are expected to be similarly distributed across different membrane domains and to exhibit similar dynamics. Fig. 1 is a schematic drawing showing structures of eye-lens lipid membranes: membranes that are close to saturation with cholesterol (Fig. 1A, lens lipid membranes from young animals and from lens cortex) and membranes that are overloaded with cholesterol (Fig. 1B, nuclear lens lipid membranes where bulk phospholipid–cholesterol domain (PCD) coexists with an immiscible pure cholesterol bilayer domain (CBD)). The phospholipid-type spin labels are expected to partition only into the bulk PCD. Thus, profiles obtained with the use of these spin labels should describe only properties of the PCD, without “contamination” from the CBD. The cholesterol-type spin labels should distribute between both domains and can detect and discriminate the PCD and the CBD (we direct readers to Ref. [11] for more details).
Section snippets
Materials
One-palmitoyl-2-(n-doxylstearoyl)phosphatidylcholine spin labels (n-PC, n = 5, 7, 10, 12, 14, or 16), tempocholine-1-palmitoyl-2-oleoylphosphatidic acid ester (T-PC), and cholesterol were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). 9-doxylstearic acid spin label (9-SASL) and cholesterol analogs, androstane spin label (ASL) and cholestane spin label (CSL) were purchased from Molecular Probes (Eugene, OR). Other chemicals, of at least reagent grade, were purchased from Sigma–Aldrich
Conventional EPR spectra
In Fig. 2, EPR spectra of selected spin labels obtained at W- and X-band for cortical lens lipid membranes are presented. Shapes of spectra indicate that phospholipid bilayers of these membranes contain high (saturating) amounts of cholesterol. This is clearly seen for the high-field component of the spectrum of 16-PC, both at X- and W-band. The shape of this line is similar to the shape of the high field component of 7- or 10-PC in membranes without cholesterol (data not shown). The
Conclusions
New capabilities in measurement of the spin–lattice relaxation time and oxygen transport parameter using SR EPR at W-band have been demonstrated in biological samples, namely, lens lipid membranes isolated from the cortical and nuclear region of the 2-year-old porcine eye. Results demonstrate that SR EPR and spin-label oximetry at W-band have the potential to be powerful tools for studying samples of small volume, ∼30 nL. Such capability could be essential to obtaining detailed T1 profiles
Acknowledgment
This work was supported by Grants EY015526, EB002052, EB001980, and EY001931 from the National Institutes of Health.
References (42)
- et al.
Effects of lutein and cholesterol on alkyl chain bending in lipid bilayers: a pulse electron spin resonance spin labeling study
Biophys. J.
(1996) - et al.
Saturation recovery EPR and ELDOR at W-band for spin labels
J. Magn. Reson.
(2008) - et al.
Spin-label oximetry at Q- and W-band
J. Magn. Reson.
(2011) - et al.
Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies
Biochim. Biophys. Acta
(2007) - et al.
Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches
Biochim. Biophys. Acta
(2008) - et al.
Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies
Biochim. Biophys. Acta
(2009) - et al.
The immiscible cholesterol bilayer domain exists as an integral part of phospholipid bilayer membranes
Biochim. Biophys. Acta
(2011) - et al.
The effect of protons or calcium ions on the phase behavior of phosphatidylserine–cholesterol mixtures
Biochim. Biophys. Acta
(1991) - et al.
Phase behavior of mixtures of cholesterol and saturated phosphatidylglycerols
Chem. Phys. Lipids
(1995) Cholesterol in bilayers of sphingomyelin or dihydrosphingomyelin at concentrations found in ocular lens membranes
Biophys. J.
(2003)
Lens plasma membrane: isolation and biochemical characterization
Exp. Eye Res.
A simple method for the isolation and purification of total lipids from animal tissues
J. Biol. Chem.
Concentration by centrifugation for gas exchange EPR oximetry measurements with loop-gap resonators
J. Magn. Reson.
High-field ESR on aligned membranes: a simple method to record spectra from different membrane orientations in the magnetic field
J. Magn. Reson.
Membrane cholesterol and phospholipid in consecutive concentric sections of human lenses
J. Lipid Res.
Age-dependent changes in the distribution and concentration of human lens cholesterol and phospholipids
Biochim. Biophys. Acta
Human lens lipids differ markedly from those of commonly used experimental animals
Biochim. Biophys. Acta
Three-dimensional dynamic structure of the liquid-ordered domain in lipid membranes as examined by pulse-EPR oxygen probing
Biophys. J.
Pulse EPR detection of lipid exchange between protein-rich raft and bulk domains in the membrane: methodology development and its application to studies of influenza viral membrane
Biophys. J.
Saturation recovery
Spin-label EPR T1 values using saturation recovery from 2 to 35 GHz
J. Phys. Chem. B
Cited by (20)
Molecular order and T<inf>1</inf>-relaxation, cross-relaxation in nitroxide spin labels
2018, Journal of Magnetic ResonanceLipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens
2014, Experimental Eye ResearchCitation Excerpt :The second value of the OTP measured with ASL is extremely low and falls into the profile across the trapped lipid domain. Although ASL discriminate the CBD in cortical and nuclear lens lipid membranes (Mainali et al., 2011b, 2012a, 2013b; Raguz et al., 2008, 2009), we have not yet been able to discriminate the CBD in intact membranes (Mainali et al., 2012a). Fig. 7A and B shows hydrophobicity profiles across the lipid bilayer portion of intact cortical and nuclear lens membranes.