EPR LINEWIDTH (T2) METHOD TO MEASURE MEMBRANE OXYGEN PERMEABILITY

点击翻译按钮获取中文摘要

It is well known that continuous-wave EPR spectra of nitroxide probes (labels) introduced into phospholipid bilayers are sensitive to molecular oxygen. However, accurate determination of oxygen broadening from these experiments is complicated by the complex shapes of EPR spectra, which are strongly influenced by anisotropic restricted motion of the probe molecules. An accurate method is presented to extract the oxygen broadening from the spectra measured with and without oxygen and at the same temperature. The method is based on a fast convolution algorithm with Levenberg-Marquardt optimization. This method was previously applied to EPR oximetry with nitroxides exhibiting rotational motion in the fast limit. It is shown that for several membrane spin probes, the oxygen broadening can be described as homogeneous; thus, a one-linewidth-parameter fitting model is appropriate. The method is applied to measure permeability profiles of model membranes composed from 1,2-dimyristoyl-sn-glycero -3-phosphocholine above and below the main phase transition. For both membrane phases, the broadening of doxyl- and sterol-type labels is found to be homogeneous, a finding consistent with the model of Heisenberg exchange between molecular oxygen and spin probes. As an example, the method is applied to study the ethanol effect on local oxygen permeability of a phospholipid bilayer. It is shown that ehanol concentrations as low as 1% (v/v) increase oxygen permeability of the bilayer. The effect is larger at the surface of the membrane than at its center, indicating that ethanol molecules interact primarily within the polar head region of the bilayer. This is an ongoing multi-year project.