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
]. Other EPR spectrometers built in the EPR Center allow SR measurements at microwave frequencies from 2 to 94 GHz [3
]. In previous papers [3
], 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 (six-month-old calf and pig [6
]), from animals of different ages (six-month-old and two-year-old cow [6
], and from different eye regions (cortex and nucleus of a two-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
]. EPR spin-labeling methods provide a unique opportunity for determining the lateral organization of lens lipid membranes including coexisting membrane domains [10
]. 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
]. 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
], X-ray, or neutron diffraction [16
] 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 two-year-old porcine cortical and nuclear membranes, which complement the published data describing properties of two-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-analogue spin labels (see 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. is a schematic drawing showing structures of eye-lens lipid membranes: membranes that are close to saturation with cholesterol (, lens lipid membranes from young animals and from lens cortex) and membranes that are overloaded with cholesterol (, 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).
Fig. 2 Panel of representative EPR spectra of phospholipid-type spin labels in cortical lens lipid membranes. Spectra were recorded at W-band and X-band at 25°C. gxx, gyy, and gzz are indicated to show effect of a high magnetic field on the position (more ...)
Schematic drawing of the cortical lens lipid membrane which is formed by phospholipid bilayer saturated with cholesterol (PCD) (A), and the nuclear lens lipid membrane with the pure cholesterol bilayer domain (CBD) in the bulk PCD (B).