THE VISUAL PIGMENTS 



condensation of retinenci with amino groups, yet favour its con- 

 densation with sulfhydryl groups.' 



INHIBITION OF REGENERATION BY SULPH-HYDRYL REAGENTS 



Because of this result wald and brown (1952) decided to investi- 

 gate regeneration in the presence of substances which react with 

 sulph-hydryl groups. Two such reagents, monoiodacetic acid and its 

 amide, were found to be without influence on the rate or extent of 

 regeneration from bleached rhodopsin solutions supplemented with 

 retinene. On the other hand, the more powerful — SH reagent, 

 /7-chloromercuribenzoate (PCMB), when in sufficient concentration, 

 inhibited regeneration completely. Since PCMB was found to be 

 without effect, either on rhodopsin itself or on retinene, it appeared 

 that PCMB must react with opsin in some way. Now PCMB reacts 

 with proteins to form a mercaptide, the mercury replacing hydrogen 

 in the — SH groups. To a limited extent mercaptide formation is a 

 reversible reaction; the — SH groups on the protein can be rein- 

 stated if another substance for which PCMB has a greater affinity is 

 added. Thus if, to a solution of bleached rhodopsin and retinene 

 which has been 'poisoned' with PCMB, glutathione is added, some 

 regeneration (e.g. 30 per cent of normal) occurs. The effect of gluta- 

 thione in reversing the inhibition is greater the sooner it is added. 

 This suggests that reaction with PCMB is followed in opsin (as in 

 other proteins) by irreversible changes akin to denaturation. 



An attempt was made, by studying the effect of different concentra- 

 tions of PCMB to ascertain how many — SH groups took part in the 

 synthesis of rhodopsin from retinene and opsin. It was found, how- 

 ever, that the lowest concentration of PCMB which completely 

 inhibited regeneration was 10-12 mole equivalent. This suggested 

 that the preparations contained many more — SH groups titratable 

 with PCMB than directly participated in the Hnkage of retinene to 

 opsin. 



liberation of SULPH-HYDRYL GROUPS WHEN RHODOPSIN 

 BLEACHES 



In order to obtain a trustworthy figure for the number of — SH 

 groups directly involved in the linkage of retinene^ to opsin, wald 

 and BROWN (1952) carried out an amperometric titration of the 

 additional — SH groups exposed when rhodopsin is bleached. 



A standard mercury-mercuric iodide half cell was connected by 



118 



