Phase-Separation and Domain-Formation in Cholesterol-Sphingomyelin Mixture: Pulse-EPR Oxygen Probing

Membranes made of Chol/ESM (cholesterol/egg sphingomyelin) mixtures were investigated using saturation-recovery electron paramagnetic resonance spin-labeling methods, in which bimolecular collisions of relaxation agents (oxygen or nickel ethylenediamine diacetic acid) with spin labels are measured. Liquid-disordered (I-d) and liquid-ordered (I-o) phases, and cholesterol bilayer domains (CBDs) were discriminated and characterized by profiles of the oxygen transport parameter (OTP). In the I-d phase, coexisting with the I-o phase, the OTP profile is bell-shaped and lies above that in the pure ESM membrane. Changes in the OTP profile across the I-o phase are complex. When the I-o phase coexists with the I-d phase, the OTP profile is similar to that across the pure ESM membrane but with a steeper bell shape. With an increase in cholesterol concentration (up to the cholesterol-solubility threshold), the profile becomes rectangular, with low OTP values from the membrane surface to the depth of C9, and high values in the membrane center. This approximately threefold increase in the OTP occurs at the depth at which the rigid ring structure of cholesterol is immersed. Further addition of cholesterol and the formation of the CBD does not affect the OTP profile across the I-o phase. OTP values in the CBD are significantly lower than in the I-o phase.