Reaction of the purple membrane with a carbodimide

Abstract Purple membrane was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.5 and 8.0. At pH 4.5, the reaction yields cross-linked bacteriorhodopsin. The cross-linking is inhibited by pretreatment of the membrane with papain, or by the presence of carbohydrazide or glycine ethyl ester in the reaction mixture. The product of the pH 8.0 reaction is not cross-linked, but it displays altered properties. Two measures of photochemical activity (light-induced change in proton binding (Δh) and decay of photointermediate M) show changes indicative of slowed proton uptake. The Δh is increased by ethyl dimethylaminopropylcarbodiimide. This increase is unaffected by pretreatment of the membrane with papain, and it is not reversed by NH 2 OH. However, the reaction is inhibited by millimolar concentrations of CaCl 2 . The altered Δh is not apparent in detergent-solubilized membranes. Ethyl dimethylaminopropyl-carbodiimide does not appear to cause a large alteration in the membrane surface charge, as measured by Ca 2+ binding. We conclude that (1) at acid pH, ethyl dimethylaminopropylcarbodiimide can be used for cross-linking or for attachment of specific probes to the C-terminal region of bacteriorhodopsin, and hence to the cytoplasmic side of the purple membrane, and (2) at alkaline pH, ethyl dimethylaminopropylcarbodiimide reacts at a different type of site and appears to inhibit the proton pump.