2l8 G. WALD VOL. 4 (1950) 



I have referred to this procedure as a chromatographic oxidation. The founder of 

 chromatography, Michael Tswett, looked forward to the discovery of an entire class 

 of such reactions, in which dry powders act at once as adsorbents and reagents, and I 

 have no doubt that this is a correct view. Such reactions probably possess a degree of 

 specificity and orientation not commonly realized in free solution, mimicking on occasion 

 the character of enzymic processes. The range and properties of such chromatographic 

 procedures deserve careful sj'stematic examination. 



THE COENZYME OF RETINENE REDUCTION* 



A simple procedure has been described for oxidizing vitamin Aj to retinenci. In 

 the retina just the reverse process occurs : retinene^ is leduced irreversibly to vitamin Aj. 



Several years ago, as noted above, we brought this reaction into a cell-free prepara- 

 tion from cattle retinas. The retinas were frozen-dried, ground to a fine powder, and 

 were extracted exhaustively with petroleum ether, all in darkness. The residue was 

 stirred into a brei with neutral phosphate buffer. On exposing this to light, its rhodopsin 

 was bleached, and the retinenCi so formed was converted almost completely to vita- 

 min Aj. 



In a study of the bleaching of rhodopsin in aqueous solution some years ago, we 

 found that freshly prepared solutions undergo a special type of bleaching, which con- 

 tinues further than the bleaching of the same solutions after a period of aging (Wald, 

 1937-38). Bliss (1948) has lately reported that the basis of this extra bleaching in 

 fresh rhodopsin solutions is the conversion of retinencj to vitamin A^. We have con- 

 firmed this observation. A fresh rhodopsin solution, however, is not a satisfactory pre- 

 paration in which to study the reduction of retinenCj, for while this reaction is in 

 progress, the enzyme system which accompHshes it is being rapidly inactivated, the 

 vitamin A^ formed is being destroyed, and the intrusion of intermediates between 

 rhodopsin and retinenCi leaves equivocal the actual substrate in the process. 



In order to analyse such systems further one would ordinarily attempt to fractionate 

 them into their components. We had already begun such experiments when the investi- 

 gation took a new turn with the discovery that the enzyme system can be fractionated 

 anatomically through the structure of the retinal rods. 



The vertebrate rod is composed of two sections, the inner and outer limbs or seg- 

 ments. The inner limb contains the nucleus, and is the principal seat of the ordinary 

 cellulai functions. The outer limb is a specialized outgrowth, which contains all the 

 rhodopsin of the retina, and includes within its small compass the whole photoreceptor 

 process. 



When a retina is removed from the eye into Ringer solution with all possible care, 

 the solution is found to contain large numbers of rod outer limbs which had broken 

 off in the course of the dissection, just at their junctures with the inner segments. By 

 scraping, one can break away about half the outer limbs from the surface of the retina, 

 and collect them in a dense suspension, free from other retinal tissues, by filtration or 

 differential centrifugation (Fig. 2). 



When this procedure is cariied out in dim red light, the outer limbs contain a 

 large quantity of rhodopsin. On exposure to white light this bleaches; but in the isolated 



* A detailed account of the experiments reviewed in this section will be found in the paper of 

 Wald and Hubbard (1948-49). 



References p. 228. 



