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Intramembrane Polarity by Electron Spin Echo Spectroscopy of Labeled Lipids

Rosa Bartucci^*, Rita Guzzi^*, Derek Marsh and Luigi Sportelli^*

^* Dipartimento di Fisica and Unità INFM, Università della Calabria, I-87036 Arcavacata di Rende (CS), Italy; and Max-Planck-Institut für biophysikalische Chemie, Abteilung Spektroskopie, 37077 Göttingen, Germany

Correspondence: Address reprint requests to Dr. R. Bartucci, Dipartimento di Fisica ed Unità INFM, Università della Calabria, I-87036 Arcavacata di Rende (CS), Italia. Tel.: +39-0984-496074; Fax: +39-0984-494401; E-mail: bartucci{at}fis.unical.it.

The association of water (D₂O) with phospholipid membranes was studied by using pulsed-electron spin resonance techniques. We measured the deuterium electron spin echo modulation of spin-labeled phospholipids by D₂O in membranes of dipalmitoyl phosphatidylcholine with and without 50 mol% of cholesterol. The Fourier transform of the relaxation-corrected two-pulse echo decay curve reveals peaks, at one and two times the deuterium NMR frequency, that arise from the dipolar hyperfine interaction of the deuterium nucleus with the unpaired electron spin of the nitroxide-labeled lipid. For phosphatidylcholine spin-labeled at different positions down the sn-2 chain, the amplitude of the deuterium signal decreases toward the center of the membrane, and is reduced to zero from the C-12 atom position onward. At chain positions C-5 and C-7 closer to the phospholipid headgroups, the amplitude of the deuterium signal is greater in the presence of cholesterol than in its absence. These results are in good agreement with more indirect measurements of the transmembrane polarity profile that are based on the ¹⁴N-hyperfine splittings in the conventional continuous-wave electron spin resonance spectrum.

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