Spin-Labeled Small Unilamellar Vesicles with the T 1-Sensitive Saturation-Recovery EPR Display as an Oxygen-Sensitive Analyte for Measurement of Cellular Respiration
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This study validated the use of small unilamellar vesicles (SUVs) made of 1-palmitoyl-2-oleoylphosphatidylcholine with 1 mol % spin label of 1-palmitoyl-2-(16-doxylstearoyl)phosphatidylcholine (16-PC) as an oxygen-sensitive analyte to study cellular respiration. In the analyte, the hydrocarbon environment surrounds the nitroxide moiety of 16-PC. This ensures high oxygen concentration and oxygen diffusion at the location of the nitroxide as well as isolation of the nitroxide moiety from cellular reductants and paramagnetic ions that might interfere with spin-label oximetry measurements. The saturation-recovery electron paramagnetic resonance approach was applied in the analysis since this approach is the most direct method to carry out oximetric studies. It was shown that this display (spin–lattice relaxation rate) is linear in oxygen partial pressure up to 100 % air (159 mmHg). Experiments using a neuronal cell line in suspension were carried out at X-band for closed-chamber geometry. Oxygen consumption rates showed a linear dependence on the number of cells. Other significant benefits of the analyte are: the fast effective rotational diffusion and slow translational diffusion of the spin-probe is favorable for the measurements, and there is no cross-reactivity between oxygen and paramagnetic ions in the lipid bilayer.
KeywordsElectron Paramagnetic Resonance Electron Paramagnetic Resonance Spectrum Nitroxide Electron Paramagnetic Resonance Signal Spin Label
This work was supported by Grants EY015526, EB002052, EB001980, and NS081936 from the National Institutes of Health.
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