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

Abstract

ABSTRACTThe analysis of membrane protein topography using fast photochemical oxidation of protein (FPOP) has been reported in recent years, but 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. Here, we investigated the effect of bulk hydroxyl radical scavenging in FPOP using both isolated cellular membranes as well as detergent micelles. We found no significant change in radical scavenging activity upon the addition of disrupted cellular membranes with the membrane concentration in the range of 0-25600 cell/μL using an inline radical dosimeter. We confirmed the non-scavenging nature of the membrane 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 acts as a potent hydroxyl radical scavenger as expected, additional detergent has little to no hydroxyl radical scavenging effect once the critical micelle concentration is reached. These results suggest that any scavenging effect of membranes or organized amphiphilic membrane mimetics in FPOP experiments are not due to bulk hydroxyl radical scavenging, but may be due to a localized scavenging phenomenon.