Further characterization of the lipid-depleted bovine rhodopsin obtained by cholate-ammonium sulfate fractionation

Abstract

The rhodopsin preparation obtained by the method of ammonium sulfate fractionation contained 3–6 mol phospholipid and about 18 mol cholate per mol rhodopsin. The purified rhodopsin had 74% helical structure and showed a visible CD spectrum different from that of rhodopsin in the membrane. The rhodopsin was stable below but denatured gradually above 20°C. The lifetime of metarhodopsin I was long in this preparation. Regeneration capacity was low and only 30% of the original rhodopsin was regenerable by addition of 11- cis-retinal after bleaching. 50 mol of phosphatidylcholine were maximally bound to 1 mol rhodopsin when the purified rhodopsin was mixed with phosphatidylcholine in 0.5% cholate. The rhodopsin recombined with lipid had properties similar to those of the original rhodopsin in the membrane. Exchange of cholate for other detergents was easily performed by dialysis. The rhodopsin preparation in which cholate was exchanged for digitonin gave almost the same CD, thermal stability and regenerability as those of a native rhodopsin in the membrane but metarhodopsin I still retained its long lifetime.