Reliability of nitroxide spin probes in reporting membrane properties: a comparison of nitroxide- and...

The reliability for the study of membrane properties of the steroid nitroxide spin probe, 3-doxylcholestane, was tested by comparison of analogous data for the deuterated steroid, cholesterol-3 alpha-d. Good agreement between the two probes was found for the dependence of their order parameters on variation of temperature or cholesterol concentration in egg phosphatidylcholine bilayers. This finding is contrasted with the results of a previous study of fatty acid probes where poor agreement was found for the spectral responses of nitroxide- and deuterium-labeled species. The angular dependence of the ESR spectra of nitroxide-labeled probes in oriented multibilayer films was examined to determine if the probes were oriented in a tilted fashion in the bilayer. The 3-doxylcholestane probe and a doxylstearic acid labeled at position 14 orient with their long molecular axes perpendicular to the bilayer plane. In contrast, the stearic acid probe nitroxide labeled at position 5 does not appear to orient in such a fashion. We suggest that the behavior of the latter probe reflects the difficulty of inserting a bulky nitroxide group into a highly ordered region of the bilayer rather than an inherent tilting of the phospholipid acyl chains. On the basis of the comparisons between various types of probes, some suggestions are made concerning the choice of ESR spin probe to obtain reliable information in membrane studies.