THE VISUAL PIGMENTS 



retinae in three or four changes of acid buffer solution (pH 4-5) all 

 the blood and most of the other contaminants can be washed away 

 without affecting the visual pigment. 



To obtain less-contaminated preparations, involved procedures 

 are necessary. These, for the most part, have been worked out for 

 the visual purple of frog or cattle retinae. There is good reason, 

 however, to suppose that the methods described below have a fairly 

 general application. 



In 1937 LYTHGOE noticed that sHght disturbance of a retina im- 

 mersed in fluid was sufficient to cause numbers of rod outer-segments 

 to break off. Since the visual purple is contained in the outer seg- 

 ments, an extract made from these alone would include all the visual 

 pigment, but none of those impurities whose source was in the other 

 structures of the retina, lythgoe, therefore, completed the process 

 of detachment by vigorously shaking some frogs' retinae with 0-6 per 

 cent salt solution. The mass was then poured through a wire gauze 

 of 200 threads to the inch. This allowed the outer segments to pass 

 through but held back the fragments of retinal debris, lythgoe then 

 centrifuged the filtrate, removed the supernatant salt solution and 

 extracted the residue with digitonin solution. By these means he 

 obtained a visual purple solution which contained less impurities 

 than one made from whole retinae. 



saito (1938) subsequently described a neater way of obtaining an 

 outer segment concentrate. The retinae are shaken with 35 or 40 per 

 cent sucrose solution. This has a specific gravity nearly the same as 

 that of the outer limbs but less than that of the pigment granules of 

 the epithelium and other parts of the retina. Consequently, when the 

 mixture is centrifuged the outer segments remain in a suspension 

 which can be withdrawn from the unwanted debris. In practice, the 

 aqueous and vitreous humours — a drop of which usually accom- 

 panies each retina — often so dilute the sucrose solution that only a 

 poor yield of outer segments is obtained in the first flotation. In 

 this event, and in any case where quantitative yields are sought, the 

 procedure may be repeated by shaking the centrifugate with a fresh 

 portion of sucrose solution. 



The suspension is then diluted (e.g. with pH 4-6 buffer solution) to 

 about three times its original volume. The outer segments start to 

 settle out, a process which may be hastened by spinning at 4,000 r.p.m. 

 for 10 minutes or so. The clear supernatant can then be withdrawn 

 and discarded, leaving the compacted mass of outer segments at the 



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