Spin-label saturation-recovery EPR at W-band: Applications to eye lens lipid membranes

https://doi.org/10.1016/j.jmr.2011.06.014Get rights and content

Abstract

Saturation-recovery (SR) EPR at W-band (94 GHz) to obtain profiles of the membrane fluidity and profiles of the oxygen transport parameter is demonstrated for lens lipid membranes using phosphatidylcholine (n-PC), stearic acid (n-SASL), and cholesterol analog (ASL and CSL) spin labels, and compared with results obtained in parallel experiments at X-band (9.4 GHz). Membranes were derived from the total lipids extracted from 2-year-old porcine lens cortex and nucleus. Two findings are especially significant. First, measurements of the spin–lattice relaxation times T1 for n-PCs allowed T1 profiles across the membrane to be obtained. These profiles reflect local membrane properties differently than profiles of the order parameter. Profiles obtained at W-band are, however, shifted to longer T1 values compared to those obtained at X-band. Second, using cholesterol analog spin labels and relaxation agents (hydrophobic oxygen and water-soluble NiEDDA), the cholesterol bilayer domain was discriminated in membranes made from lipids of the lens nucleus. However, membranes made from cortical lipids show a single homogeneous environment. Profiles of the oxygen transport parameter obtained from W-band measurements are practically identical to those obtained from X-band measurements, and are very similar to those obtained earlier at X-band for membranes made of 2-year-old bovine cortical and nuclear lens lipids (M. Raguz, J. Widomska, J. Dillon, E.R. Gaillard, W.K. Subczynski, Biochim. Biophys. Acta 1788 (2009) 2380–2388). Results demonstrate that SR EPR at W-band has the potential to be a powerful tool for studying samples of small volume, ∼30 nL, compared with the sample volume of ∼3 μL at X-band.

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)

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