Studies in rhodopsin. 4. Preparation of rhodopsin

Abstract

Various methods for the separation of rod outer segments from cattle retinas were investigated, attention being paid to the optical purity of the rhodopsin solns. obtained by extraction of the rod outer-segments. A new method was developed depending on the fact that cattle rods have the same density as 0.88 [image] sucrose. The rods are separated by centrifuging them in a sucrose soln. in which a density gradient has been established. Various treatments of the isolated rods were tried in order to eliminate impurities. The best results have been obtained by treating with 4% (wt./vol.) alum soln. for 1 hr. followed by 40% (wt./vol.) neutralized formaldehyde soln. for 10 min. The effects of these various treatments on the appearance of the rods was followed microscopically. Very pure cattle rhodopsin solns. were obtained showing maxima at 498, 345 and 275 m[mu]. The last band can be accounted for by the presence of tyrosine and tryptophan only. Making some plausible assumptions, rhodopsin appears to contain 6% tyrosine 3% tryptophan on the dry weight of the protein.