Determining radical penetration into membranes using ESR splitting constants

Abstract

Determination of the depth of radical penetration into a lipid membrane is critical to the understanding of the role membranes play in radical attack. We have previously studied radical penetration into lipid bilayers using novel lipophilic spin traps and a combination of NMR and ESR techniques. We now focus on erythrocyte ghost (EG) membranes. Based on a correlation between ESR β-H splitting constants (a β-H) and solvent polarity, we have been able to locate stable radicals such as doxyls 2–4 and spin adducts 6–8 intercalated within liposomal bilayers and EG membranes. As a rule, the more lipophilic a spin adduct, the deeper it is found in the bilayer; however, the depth of penetration also depends on the steric bulk of the intercalant and whether intercalation is effected by sonication or diffusion, with the former more energetic and more effective. Compared to simple liposomes, the head group region of the red blood cell membrane is more rigid and lipophilic because of the presence of cholesterol. Hence, the biomembrane head group filters out possible intercalants that are not sufficiently lipophilic. Steric bulk plays less of a role in the EG system, perhaps because the cholesterol introduces a greater element of disorder, attenuating the role played by lipid–lipid interactions.