Partial separation of inwardly pumping and outwardly pumping bacteriorhodopsin reconstituted liposomes by gel filtration

Abstract

Reconstitution of bacteriorhodopsin using the reverse-phase evaporation technique leads to the formation of both inwardly proton pumping and outwardly proton pumping liposomes [(1985) FEBS Lett. 188, 101–1061]. The reconstituted preparation was size fractionated using gel filtration chromatography on Sephacryl S1000. The direction of proton pumping was assessed in various fractions using the pH sensitive fluorescence probe pyranine. The proportion of outwardly pumping liposomes was shown to increase with decreasing liposome size. The larger liposomes (diameter larger than 200 nm) were shown to be pure inwardly pumping liposomes with almost homogeneous bacteriorhodopsin orientation.