Self-Organized Amphiphiles Are Poor Hydroxyl Radical Scavengers in Fast Photochemical Oxidation of Proteins Experiments.

Abstract

Analysis of membrane protein topography using fast photochemical oxidation of proteins (FPOP) has been reported in recent years but is still underrepresented in literature. Based on the hydroxyl radical reactivity of lipids and other amphiphiles, it is believed that the membrane environment acts as a hydroxyl radical scavenger decreasing effective hydroxyl radical doses and resulting in less observed oxidation of proteins. We found no significant change in bulk solvent radical scavenging activity upon the addition of disrupted cellular membranes up to 25600 cells/μL using an inline radical dosimeter. We confirmed the nonscavenging nature of the membrane in bulk solution with the FPOP results of a soluble model protein in the presence of cell membranes, which showed no significant difference in oxidation with or without membranes. The use of detergents revealed that, while soluble detergent below the critical micelle concentration is a potent hydroxyl radical scavenger, additional detergent has little to no hydroxyl radical scavenging effect once the critical micelle concentration is reached. Examination of both an extracellular peptide of the integral membrane protein bacteriorhodopsin as well as a novel hydroxyl radical dosimeter tethered to a Triton X-series amphiphile indicate that proximity to the membrane surface greatly decreases reaction with hydroxyl radicals, even though the oxidation target is equally solvent accessible. These results suggest that the observed reduced oxidation of solvent-accessible surfaces of integral membrane proteins is due to the high local concentration of radical scavengers in the membrane or membrane mimetics competing for the local concentration of hydroxyl radicals.