VOL. 4 (1950) 



RETINENES AND VITAMINS A 



223 



ethanol as compared with hexane (cf. Wald, 1947-48). That the carbonyl group replaces 

 the primary alcohol group of vitamin A, is shown by the fact that though the vitamin 

 is hypophasic, its oxidation product is epiphasic in partition between hexane and 90% 

 methanol. This information, together with what follows, leaves little doubt that this 

 product is retinene.,, and that it is the aldehyde of vitamin A2. 



As in the manufacture of retinene 1, we have found that the oxidation of vitamin A2 

 to retinene.j can be carried out conveniently in chromatographic form. The procedure 

 is identical with that used in making retinencj ; but in this case only about half as much 

 manganese dioxide is employed — 0.3 g to oxidize 10 mg of vitamin A,. The yield of 

 retinenca is in the neighbourhood of 50% ; and it can be brought to a state of high purity 

 by chromatographic adsorption on a column of calcium carbonate. 



In our past experience one of the most remarkable properties of the porphyropsin 

 system has been its detailed parallelism in chemical behaviour with the rhodopsin 

 cycle. In the present instance this parallelism is maintained, for retinencj is reduced to 

 vitamin A., by an enzyme system entirely similar to that which reduces retinene^. 



This system can be assembled from the following components: the coenzyme, 

 DPN-Ho; the substrate, sjmthetic retinene^, prepared by the chromatographic oxidation 

 of vitamin A;, on manganese dioxide; and the apoenzyme, contained in a clear, almost 

 colourless saline extract of homogenized freshwater fish retinas (yellow perch, sunfish). 

 When these three components are mixed and left at room temperature for two hours, 

 the retinene., is reduced almost entirely to vitamin A.^ (Fig. 7, upper half). 



Since the coenzyme of retinene reduction is common to the rhodopsin and por- 

 phyropsin cycles, one may inquire into the specificity of the apoenzyme. To test this, 

 experiments were performed in which the frog apoenzyme was allowed to act on retinene.^ 

 and the fish apoenzyme on retinene j . It emerged that the reduction proceeded as smooth- 

 ly and completely with the crossed as with the normal substrates (Figs 6 and 7). 



There is no reason therefore to designate the apoen- 

 zyme differently in the rhodopsin and porphyropsin § 

 systems. We have to deal with a single apoenzyme, |o.j 

 retinene reductase, which with the single coenzyme, i 

 dihydrocozymase, reduces either of the retinenes to the o.z 

 corresponding vitamin A. 



This enzyme system introduces a new vitamin into a, 

 the chemistry of rod vision, for the central component 



Fig. 7. Action of retinene reductase from a freshwater fish on 

 synthetic retinencj and retinenej. The experimental mixtures 

 included solutions of the retinenes in 1% digitonin, 2.4 mg 

 reduced cozymase per ml, 6-7 mg nicotinamide and i mg 

 a-tocopheryl phosphate per ml to stabilize the system; and 

 extracts of homogenized yellow perch retinas in m/30 phosphate 

 buffer, ph 6.81. The controls differed only in that the retinal 

 extracts were replaced by the phosphate buffer alone. All the 

 mixtures were left for 2 hours at 22° C; then methanol was 

 added to a concentration of 60%, and they were extracted 

 vWth hexane. The spectra of the hexane extracts are shown. 

 Those from the controls (solid circles) show the unaltered 

 retinenes; those from the enzyme mixtures (open circles) show 

 almost complete conversion to the corresponding vitamins A. 

 In each figure a short vertical line shows the position of the 

 absorption maximum of vitamin Aj or .\j in hexane. 



References p. 228. 



0.3 



300 20 40 60 eO MO 70 M 60 80 

 Wavelength -mji 



