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

Abstract

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.