THE VISUAL PIGMENTS 



to form rhodopsin in good yield. Opsin was prepared, e.g. from 

 cattle retinae, in the following way. The retinae were removed and 

 bleached to the colourless condition by exposure to light. The outer 

 segments of the rods were then separated from the rest of the retina 

 by flotation in 45 per cent sucrose solution. This was then diluted. 

 The bleached outer segments were centrifuged out, washed, and 

 extracted with 2 per cent digitonin solution. 



The synthesis of rhodopsin from retinenci and opsin is inhibited 

 by 0-1 M hydroxylamine. This is because hydroxylamine condenses 

 with retinenci to form an oxime, and thus acts in competition with 

 opsin for the aldehyde group in the retinenci molecule. The syn- 

 thesis of rhodopsin is likewise inhibited by 0-7 M formaldehyde. In 

 this case, however, the aldehyde group of formaldehyde competes 

 with that of retinenci for those groups in the opsin molecule which 

 are involved in the reaction. 



Now the work of morton and his colleagues had suggested that 

 in the formation of indicator yellow, and hence of rhodopsin, reti- 

 nene reacts with an amino group. However, aldehydes will also 

 readily react with sulph-hydryl ( — SH) groups, wald and brown 

 (1952) therefore proceeded to consider which of these alternative 

 reactions would provide a basis for the synthesis of rhodopsin. 



In solutions more acid than pH 6-7, amino groups exist mainly in 



the form of ammonium ions ( — NHg), which do not readily react 

 with aldehydes. Thus morton and co-workers had found that the 

 reaction between aliphatic amino compounds and retinene took place 

 only in alkaline solution (p. 105), and even then only slowly unless 

 the amino compound was present in excess. Sulph-hydryl groups, on 

 the other hand, remain unionized up to about pH 8-9 and only in 

 more alkaline solutions are they ionized ( — S~). At lower pH's, sulph- 

 hydryl groups readily react with formaldehyde and other aldehydes. 

 WALD and BROWN therefore investigated the regeneration of 

 rhodopsin, from retinenci and opsin, over a wide pH range. In their 

 experiments 0-5 ml of a rhodopsin solution was mixed in the dark 

 with 0-25 ml of a concentrated solution of retinenci in digitonin and 

 with 0-25 ml of buff"er or another reagent. A sample of the solution 

 was then completely bleached by exposing it to a 160 W tungsten 

 lamp, suitable filters being used to remove heat and ultra-violet 

 radiation. The bleached solution was then allowed to regenerate in 

 darkness for 2-2 J hr at 24-27°C. At the end of this period 0-1 ml of 



116 



