An Electron Paramagnetic Resonance Method for Measuring the Affinity of a Spin-Labeled Analog of Cholesterol for Phospholipids

作者：Williams Justin A; Wassall Cynthia D; Kemple Marvin D; Wassall Stephen R^*

来源：Journal of Membrane Biology, 2013, 246(9): 689-696.

DOI：10.1007/s00232-013-9586-z

• Partition coefficient
• Cholesterol
• Spinlabeled cholestane
• Cyclodextrin
• Large unilamellar vesicle
• Unsaturated lipid

Cholesterol (chol)-lipid interactions are thought to play an intrinsic role in determining lateral organization within cellular membranes. Steric compatibility of the rigid steroid moiety for ordered saturated chains contributes to the high affinity that holds chol and sphingomyelin together in lipid rafts whereas, conversely, poor affinity of the sterol for highly disordered polyunsaturated fatty acids (PUFAs) is hypothesized to drive the formation of PUFA-containing phospholipid domains depleted in chol. Here, we describe a novel method using electron paramagnetic resonance (EPR) to measure the relative affinity of chol for different phospholipids. We monitor the partitioning of 3 beta-doxyl-5 alpha-cholestane (chlstn), a spin-labeled analog of chol, between large unilamellar vesicles (LUVs) and cyclodextrin (m beta CD) through analysis of EPR spectra. Because the shape of the EPR spectrum for chlstn is sensitive to the very different tumbling rates of the two environments, the ratio of the population of chlstn in LUVs and m beta CD can be determined directly from spectra. Partition coefficients () between lipids derived from our results for chlstn agree with values obtained for chol and confirm that decreased affinity for the sterol accompanies increasing acyl chain unsaturation. The virtue of this EPR method is that it provides a measure of chol binding that is quick, employs a commercially available probe and avoids the necessity for physical separation of LUVs and m beta CD.

• 出版日期2013-9