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

Abstract

The presence of integral membrane proteins induces the formation of distinct domains in the lipid bilayer portion of biological membranes. Qualitative application of both continuous wave (CW) and saturation recovery (SR) electron paramagnetic resonance (EPR) spin-labeling methods allowed discrimination of the bulk, boundary, and trapped lipid domains. A recently developed method, which is based on the CW EPR spectra of phospholipid (PL) and cholesterol (Chol) analog spin labels, allows evaluation of the relative amount of PLs (% of total PLs) in the boundary plus trapped lipid domain and the relative amount of Chol (% of total Chol) in the trapped lipid domain [M. Raguz, L. Mainali, W. J. O’Brien, and W. K. Subczynski (2015), Exp. Eye Res., 140:179–186]. Here, a new method is presented that, based on SR EPR spin-labeling, allows quantitative evaluation of the relative amounts of PLs and Chol in the trapped lipid domain of intact membranes. This new method complements the existing one, allowing acquisition of more detailed information about the distribution of lipids between domains in intact membranes. The methodological transition of the SR EPR spin-labeling approach from qualitative to quantitative is demonstrated. The abilities of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses. Statistical analysis (Student’s t-test) of the data allowed determination of the separations of mean values above which differences can be treated as statistically significant (P ≤ 0.05) and can be attributed to sources other than preparation/technique.

Detection of cholesterol bilayer domains in intact biological membranes: Methodology development and its application to studies of eye lens fiber cell plasma membranes

“…Both spin labels work well in model membranes (Mainali et al, 2013b; Raguz et al, 2011a, b). However, because of the very high hydrophobicity of CSL, intact membranes can be labeled only with ASL, and this spin label has been intensively used to investigate localizations and amounts of Chol in domains present in intact membranes (Mainali et al, 2017a; Mainali et al, 2012; Raguz et al, 2014, 2015b, c). Measurements with model membranes indicated that conventional EPR spectra and T 1 s measured with ASL in CBDs and surrounding PLs are similar (almost identical) and indistinguishable by conventional and SR EPR approaches (Raguz et al, 2011a, b).…”

“…2.3). However, we were very successful when using an approach, we recently applied in a similar situation (Mainali et al, 2017a), namely fixing a few parameters before applying the triple-exponential fitting. Here, we fixed the relaxation characteristics of ASL coming from the trapped lipid domain obtained from the double-exponential fitting.…”

“…SR EPR signals were obtained by short-pulse (300 ns) experiments with 10 5 –10 6 decays acquired with 2,048 data points on each signal (decay). Measurements were performed at X-band on a home-built spectrometer with recent major hardware improvements and equipped with a loop-gap resonator (LGR) (Mainali et al, 2017a). These improvements were essential to obtaining reliable SR signals with double- and triple-exponential content.…”

“…In the previous paper (Mainali et al, 2017a), we described an optimization process for similar samples (intact cortical and nuclear fiber cell membranes isolated from two-year-old porcine and containing ASL) for deoxygenated conditions. Here, we used a similar optimization process for samples equilibrated with 30% and 50% air.…”

“…To solve our problem, we applied an approach that we have used previously (Mainali et al, 2017a), allowing us to reliably fit SR signals to triple exponentials by fixing some of the time constants. Here, we fixed T 1 (Air, TRAP)* (obtained from fitting the SR signal to a double exponential curve) and again fitted the SR signal to triple exponentials with the now fixed time constant.…”