The fidelity of response by nitroxide spin probes to changes in membrane organization: The condensing effect of cholesterol

Abstract

The response of doxyl fatty acid spin probes in egg lecithin bilayers to added cholesterol is compared with results from 2H-NMR. Large differences are found between the profiles of order parameter vs. label position and cholesterol concentration. At constant cholesterol content, the ESR spin probe order parameter decreases continuously as the label position is moved toward the terminal methyl region of the bilayer whereas an order parameter ‘plateau’ is observed for the upper region of the bilayer by 2H-NMR. In addition, the spin probe order parameters are smaller than those observed by 2H-NMR. Differences are also observed in the profiles of order parameter vs. cholesterol content for each label position. The spin probes detect a maximal response to added cholesterol for the central portion of the chains with much weaker responses near both ends of the chains. In contrast, the 2H-NMR results indicate a large, approximately constant response for the first ten positions in the chains with a decreasing response toward the terminal methyl group. For all the positions examined, the spin probes show a weaker response than that observed by 2H-NMR. A direct measure of the perturbing effect of a spin label is made by comparing the deuterium quadrupole splittings in egg lecithin-cholesterol bilayers for stearic acid with and without an attached doxyl moiety. The spin-labelled fatty acid has a much reduced quadrupole splitting and an opposite response to cholesterol addition.