Mainali Laxman, Camenisch Theodore G, Hyde James S, Subczynski Witold K
Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI USA.
Appl Magn Reson. 2017 Dec;48(11-12):1355-1373. doi: 10.1007/s00723-017-0921-x. Epub 2017 Jul 22.
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 []. 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 -test) of the data allowed determination of the separations of mean values above which differences can be treated as statistically significant ( ≤ 0.05) and can be attributed to sources other than preparation/technique.
整合膜蛋白的存在会诱导生物膜脂质双层部分形成不同的结构域。连续波（CW）和饱和恢复（SR）电子顺磁共振（EPR）自旋标记方法的定性应用能够区分主体、边界和捕获的脂质结构域。一种基于磷脂（PL）和胆固醇（Chol）类似物自旋标记的连续波EPR光谱的最新方法，可以评估边界加捕获脂质结构域中PL的相对含量（占总PL的百分比）以及捕获脂质结构域中Chol的相对含量（占总Chol的百分比）。在此，我们提出了一种基于SR EPR自旋标记的新方法，该方法能够对完整膜中捕获脂质结构域内PL和Chol的相对含量进行定量评估。这种新方法对现有方法进行了补充，能够获取有关完整膜中脂质在不同结构域间分布的更详细信息。展示了SR EPR自旋标记方法从定性到定量的方法转变。通过猪眼晶状体完整的皮质和核纤维细胞质膜说明了该方法的能力。对数据进行统计分析（Student's检验）可确定平均值的差异，高于此差异可被视为具有统计学意义（≤0.05），并且可归因于制备/技术以外的来源。
