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Comparison of CW-EPR and ESEEM technique for determination of water permeability profile in liposome membranes

Hydrophobicity profile across the membrane is an important parameter of biological membranes as it defines the barrier for permation of polar molecules into the cell interior and of stability of transmembrane proteins. In this work hydrophobicity profile was measured by conventional continous wave electron paramagnetic resonance (CW-EPR) and by electron spin echo envelope modluation (ESEEM) in two liposome suspensions with different ionic strength in order to compare the sensitivity of both methods for determination of water penetration depth into the liposome membrane. Multilamellar liposomes from egg-phosphatidylcholine, cholesterol and dicetyl phosphate were prepared in deuterium oxide, buffered with phosphate buffered saline with or without NaCl at the phosphate concetrations 0.1 or 0.01 M. Liposomes were spin labeled with stearic acid spin probes with nitroxide group at different positions on the acyl chain. Deuteruim penetration depth was measured by ESEEM technique and the signal was analyzed in the frequency domain (Fourier transform (FT)-ESEEM). The ratio of the intensities of FT-ESEEM lines at deuterium and hydrogen Larmor frequencies was used as a measure of the water penetration depth. In was also indirectly determined from the hyperfine splitting constants in frozen and in fluid state, measured by CW-EPR, as they depend on the polarity of the nitroxide droup environment. The comparisom of thwo techniques demonstrated that FT-ESEEM is more sensitive to the changes on the surface of the membrane and thus more appropriate for determination of hydrophobic barrier as the conventional CW-EPR method.

Spin labeling; Liposome; Phospholipid; Pulse EPR; ESEEM

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