Reversible and irreversible bleaching of rhodopsin in detergent solutions.

Most investigators studying rhodopsin in solution have used digitonin as the solubilizing agent. In solutions of this detergent, one can observe the bleaching of rhodopsin, as well as the reversal of bleaching by resynthesis from 11 cis retinal and opsin (e.g. see ref. 1) or by photoreversal from the intermediates of bleaching (e.g., see ref. 2). Other detergents can also solubilize rhodopsin (for review see ref. 3), but little information is available about bleaching and reversal of bleaching in solutions of these detergents. In an early study, Chase and Smith4 reported that rhodopsin did not regenerate in solutions of sodium deoxycholate, as it did in solutions of mixed bile salts (of unstated composition) or digitonin. The present paper describes the bleaching of rhodopsin in another detergent that prevents regeneration, cetyltrimethyl ammonium bromide (CH3-(CH2)15-N+-(CH3)3Br-, CTAB), and contrasts the behavior of rhodopsin in CTAB with that in digitonin. CTAB was chosen because of Bridges' reports that the corresponding chloride is an efficient solubilizing agent in which rhodopsin can be stored without appreciable loss of the pigment. The presence of 1 M sodium chloride in digitonin solutions retarded the bleaching of rhodopsin in the experiments to be described. Under these conditions, the photoreversal of bleaching could be observed during steady illumination at room temperature. In comparable CTAB solutions, however, rhodopsin bleaching was much more rapid, and no reversal of bleaching was observed either during or after illumination. Materials and Methods.-Preparation of solutions: A special effort was made to avoid treatment of the retina with protein denaturants such as alum or acids in the course of the purification procedure. Thus any difference of rhodopsin bleaching or reversal of bleaching in digitonin and in CTAB would have to be caused by the detergent alone, and not by action of the detergent on an already modified molecule. The retinas of large, dark-adapted bullfrogs (Rana catesbiana, about 500 gm) were removed by the method of Lythgoe5 and shaken in a cold, 0.9% solution of sodium chloride. The resulting slurry was filtered through 70-mesh wire gauze. Centrifugation of the filtrate for 10 min at 3200 X g sedimented the rods and retinal fragments, which were separated by Saito's method of flotation in 40% (w/v) sucrose in distilled water.6 The suspended rods were sedimented, after dilution with 2 vol of distilled water, by 30to 45-min centrifugation at 12,800 X g. The pellet was then washed once with 0.9% sodium chloride and suspended in the appropriate detergent solution. Digitonin was dissolved by boiling the detergent in distilled water (2% w/v) and cooling the solution to 0C. The rods were dispersed in the solution at 0C for 2 hr, at which time the suspension was diluted with an equal volume of 0.2 M sodium phosphate buffer (pH 7.1 at 0.1 M) and centrifuged for 20 min at 12,800 X g. Even after 2 hr, though, the remaining pellet of material was still intensely colored with rhodopsin that had not been solubilized (cf. ref. 7, p. 820). CTAB solutions were 3% (w/v) in 0.1 M sodium phosphate buffer, pH 7.1, 220C. When the rods were first dispersed in the solution, the suspension appeared slightly cloudy under red light, but cleared almost immediately; at this point the rods apparently had been dissolved. Centrifugation of the solution yielded only a trace of sediment with no rhodopsin color. In several experiments a preparation of rods was first treated with digitonin as described above, and then suspended in CTAB; as much as four tenths of the total rhodopsin (measured in units of optical