Pitfalls in the use of commercial nonionic detergents for the solubilization of integral membrane proteins: Sulfhydryl oxidizing contaminants and their elimination

Abstract

For studies of isolated integral membrane proteins, nonionic detergents are an essential class of chemicals. A serious handicap, however, is that purified detergents which have been chemically analyzed are not available commercially. One type of contaminant that complicates studies of membrane-bound proteins acts as a sulfhydryl oxidizing agent. We have developed a convenient spectrophotometric assay to detect sulfhydryl oxidizing agents and have examined a number of nonionic detergents frequently used in biochemical studies. The method involves incubation of 1% detergent solutions with a known amount of dithiothreitol for 20 h followed by back-titration of unreacted dithiothreitol with 5,5′-dithiobis(2-nitrobenzoic acid). Many of the detergents assayed were found to contain significant amounts of oxidizing agents. The amount varied between different lots of the detergent and increased upon standing in aqueous solution, presumably due to peroxide formation initiated by light and oxygen. A method for purifying commercial nonionic detergents is proposed in which sulfhydryl oxidizing contaminants are eliminated by reaction with sodium borohydride followed by silica gel column chromatography and passage through a mixed bed ion-exchange resin.