Detergent removal during membrane reconstitution

Abstract

Efficiency of detergent removal during the course of several different procedures for membrane protein reconstitution was examined. Reconstitution methods studied include ethanol injection-dialysis, detergent dialysis and detergent-gel filtration. In the ethanol injection-dialysis method, approx. 70 molecules of ethanol per 1000 molecules of phospholipid are retained even after extensive (150 h) dialysis. Efficiency of detergent removal by dialysis depends upon the detergent. However, even for sodium deoxycholate, a detergent possessing a large critical micelle concentration, there are approx. 7 molecules of deoxycholate per 1000 molecules of phospholipid retained by the bilayer even after extensive (310 h) dialysis. Detergent removal by gel filtration (Sephadex G-200 or G-50) of deoxycholate, cholic acid and Triton X-100 is more efficient than removal by dialysis; as few as 10 molecules of deoxycholate are retained per 1000 molecules of phospholipid after one column passage, taking only a few hours. Ethanol was less efficiently removed by one passage over a Sephadex column than by extensive dialysis. Removal of Triton X-100 by passage over, or dialysis against, Biobeads SM-2 resulted in a similar level of detergent retention to that found by passage over Sephadex G-200 or G-50. Utilizing gel-filtration techniques, we have examined the competition for the hydrophobic peptide of glycophorin, T(is), between sodium deoxycholate and a series of phospholipids as a possible means of obtaining a quantitative measure of protein-lipid affinity. On the basis of these preliminary studies we conclude that the T(is) peptide has a relative lipid affinity of phosphatidylinositol > phosphatidylcholine > phosphatidylserin .