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Laxman Mainali, Marija Raguz, Witold K. Subczynski; A Novel EPR Approach to Studying Samples of Small Volume: Application to Eye-Lens Lipid Membranes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3038.
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© ARVO (1962-2015); The Authors (2016-present)
To discriminate membrane domains and obtain profiles of membrane properties across these domains in porcine lens lipid membranes and to demonstrate feasibility that these experiments can be performed for samples prepared from human eyes from a single donor.
Saturation-recovery electron paramagnetic resonance (SR EPR) spectroscopy was used to study lens lipid membranes derived from the cortex and nucleus of two-year-old pig-eye lenses. Using cholesterol analogue spin labels, membrane domains were discriminated. With phospholipid-type spin labels, profiles of the membrane fluidity (spin-lattice relaxation time, T1) and oxygen transport parameter (oxygen diffusion-concentration product) across each domain were obtained. Results were acquired using SR EPR at W-band with a sample volume of 30 nL and were compared with results obtained at X-band with a sample volume of 3 μL.
The cholesterol bilayer domain (CBD) was discriminated and characterized in membranes made from nuclear lipids. Membranes made from cortical lipids showed a single homogeneous environment. Membrane fluidity of the bulk cholesterol-phospholipid domain, which surrounds the CBD, increased toward the membrane center, and profiles for the cortical and nuclear lens lipid membranes were nearly identical. These profiles changed gradually with membrane depth. Profiles of the oxygen transport parameter across this domain showed an abrupt change between the C9 and C10 positions, which is where the steroid ring structure of cholesterol reaches into the membrane. The oxygen transport parameter across the CBD was very low, indicating that this domain can form a barrier to oxygen transport.
SR data obtained in parallel at X- and W-band show that coexisting membrane domains can be discriminated and characterized by SR experiments at W-band. Also, profiles of membrane fluidity and the oxygen transport parameter obtained at X- and W-band contain the same information about membrane dynamics. A significant (and positive) difference is that the sample volume at W-band is about 100 times smaller than that at X-band. Results demonstrate that SR EPR at W-band has the potential to be a powerful tool to study samples of small volume (~30 nL) and also demonstrate that it is feasible to obtain complete information about the structure and dynamics of lens lipid membranes for samples prepared from the human eyes of a single donor.
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