Saturation Recovery EPR Spin-Labeling Method for Quantification of Lipids in Biological Membrane Domains
- Autores: Mainali L.1, Camenisch T.1, Hyde J.1, Subczynski W.1
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Afiliações:
- Department of Biophysics, Medical College of Wisconsin
- Edição: Volume 48, Nº 11-12 (2017)
- Páginas: 1355-1373
- Seção: Original Paper
- URL: https://journals.rcsi.science/0937-9347/article/view/247864
- DOI: https://doi.org/10.1007/s00723-017-0921-x
- ID: 247864
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Resumo
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 (Raguz et al. Exp Eye Res 140:179–186, 24). 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.
Sobre autores
Laxman Mainali
Department of Biophysics, Medical College of Wisconsin
Email: subczyn@mcw.edu
Estados Unidos da América, 8701 Watertown Plank Road, Milwaukee, WI, 53226
Theodore Camenisch
Department of Biophysics, Medical College of Wisconsin
Email: subczyn@mcw.edu
Estados Unidos da América, 8701 Watertown Plank Road, Milwaukee, WI, 53226
James Hyde
Department of Biophysics, Medical College of Wisconsin
Email: subczyn@mcw.edu
Estados Unidos da América, 8701 Watertown Plank Road, Milwaukee, WI, 53226
Witold Subczynski
Department of Biophysics, Medical College of Wisconsin
Autor responsável pela correspondência
Email: subczyn@mcw.edu
Estados Unidos da América, 8701 Watertown Plank Road, Milwaukee, WI, 53226