III. BIOCHEMICAL SYSTEMS 73 



liigh couccutratioii.s of retineiic, they regenerate up to 85 % after bleaching."*^ 

 What is more, one can extract from Hght-adapted rods a colorless, caro- 

 tenoid-frce solution of opsin, which on incubation with retinene in the dark 

 forms a high concentration of rhodopsin (Fig. 18). 



No other molecules than retinene and opsin are required for this reaction, 

 nor does it require an external source of energy. This is a spontaneous — i.e., 

 an energy-yielding — ^reaction. It is the bleaching of rhodopsin, its cleavage 

 into retinene and opsin, that requires energy, usually in the form of light. 

 Given the opportunity — i.e., removal of light — retinene and opsin reunite 

 spontaneously to regenerate the visual pigment. 



There remains the more difficult problem, the synthesis of rhodopsin 

 from vitamin A and opsin. No such synthesis had been observed in vitro, 

 and all attempts to stimulate it outside the living tissues had failed. Yet 

 the ease with which retinene and opsin unite to form rhodopsin suggested 

 that, if it were only possible to oxidize vitamin A to retinene, the job would 

 be done. This proved to be true. All the difficulty in making rhodopsin 

 from vitamin A is implicit in the difficulty of oxidizing vitamin A to 

 retinene. 



We have already' noted that the equilibrium between vitamin A and 

 retinene favors reduction rather than oxidation. In the much more un- 

 balanced equilibrium between ethanol and acetaldehyde, however, it has 

 long been known that the reaction can be driven in the oxidative direction 

 by introducing an aldehyde-trapping reagent — some substance, such as 

 cyanide, bisulfite, or semicarbazide, which by condensing with the aldehyde 

 removes it from the system, so forcing its continuous production. 



As a trapping reagent for retinene in the alcohol dehydrogenase system, 

 we have introduced hydroxylamine, NHoOH. This condenses rapidly with 

 retinene at room temperature to form retinene oxime: 



C19H27CHO -I- NH2OH ^ Ci9H27CH=NOH -I- II2O 

 Retinene -|- hydroxylamine Retinene oxime 



In the presence of this reagent, the alcohol dehydrogenase system ox- 

 idizes vitamin A to retinene in considerable yield .^^ 



The retina, however, already contains the specific retinene-t rapping 

 reagent, opsin. Opsin should be able to substitute in the alcohol dehj'drogen- 

 ase system for hydroxylamine and should drive a continuous oxidation of 

 vitamin A to retinene, bj^ continuously removing retinene to form rhodop- 

 sin. 



The trouble with this notion is that an isolated retina, bleached to color- 

 lessness, and incubated in the dark, seems to contain all the components 



" G. Wald and R. Hubbard, Proc. Natl. Acad. Sci. U. S. 36, 92 (1950). 



