Monolayer Film of Rhodopsin at the Air/Water Interface

Abstract

MANY reports have been made of chemical and physical properties of rhodopsin^1,2. A role of rhodopsin in visual excitation has been fully discussed by Wald^3,4. However, most of the work so far has been carried out using aqueous solutions of rhodopsin extracted with digitonin, resulting in a digitonin complex. Accordingly, there is a risk of observing reactions of rhodopsin different from those primarily found in vivo. Recently, electron microscopic findings on the ultra-fine structure of visual cells^5,6 suggest that rhodopsin may be located between the double layers of the disk-like units of rod outer-segment which forms a monolayer. It seems useful to pursue the physico-chemical properties of rhodopsin monolayer in vitro in order to postulate models for the molecular function of rhodopsin. Weitzel et al.⁷ reported properties of the monolayer of vitamin A and its derivatives spread on aqueous solution. Many workers since Devaux⁸ have investigated the properties of protein films spread on water. We have succeeded in preparing the monolayer film of rhodopsin—vitamin A₁ aldehyde combined with protein— at the air/water interface, and have measured the changes of surface pressure following illumination. An extracted rhodopsin solution was dropped on a clean aqueous surface with a micropipette as was the case with spreading a monolayer of protein. The substrate surmounted with rhodopsin consisted of a phosphate buffer solution having pH of 6.8. The surface pressure exerted on the film was measured with a modified method of Sasaki⁹ under dim red light of wave-length longer than 750 mµ. To plot the film pressure against the area of the spread films, a barrier placed at a distance of 40 cm. from the other fixed barrier was slid on a trough at a constant speed with certain intervals until the distance was reduced to about 25 cm. Thus the plotting of the film pressure yielded a force-area curve. Besides the force-area curve, changes of surface pressure were measured at any area. In the meantime the barrier was kept unslid so that the rhodopsin film might reveal an unstable surface pressure. A rhodopsin solution extracted with diluted ethanol reported by Hosoya et al.¹⁰ seemed to be the most suitable agent in preparing a monolayer film. The digitonin extract did not seem to be satisfactory in monolayer experiments, because of the surface activity of digitonin and higher molecular weight of digitonin complex; but it might be observed in comparison with ethanol extract. Fig. 1 shows the force-area curve of the monolayer film of rhodopsin extracted with diluted ethanol. At a low-pressure region, surface pressure increased slowly as the area was diminished.