VOL. 4 (1950) RETINENES AND VITAMINS A 225 



enzyme system. This was solved initially by bringing the retinenes into water solution 

 with the aid of digitonin, with which they form water-sduble complexes. 



The use of digitonin, however, proved to be unnecessary. The retinal extracts which 

 contain the apoenzyme take up the retinenes directly. If either of the retinenes is 

 concentrated in a few drops of petroleum ether, and is agitated together with a water 

 extract of retinas while the last of the petroleum ether is drawn off under suction, the 

 retinenes gradually are taken up to yield clear yellow solutions. This is one indication 

 that the retinenes couple with water-soluble substances from the retina. Primarily in 

 these preparations they attach to protein, for they are precipitated from such solutions 

 with the protein fraction. 



It has been known for some time that in the product of bleaching rhodopsin in 

 solution, most of the retinenej is found loosely coupled with protein (Wald, 1937-38, 

 pp. 812-813). In this condition it behaves as a pn indicator, deep yellow in acid and 

 almost colourless in alkaline solution; hence Lythgoe's proposal that it be called 

 "indicator yellow". Synthetic retinenei does not change its spectrum at all with pn; 

 nor does natural letinene^ after partial purification by adsorption and elution (Wald, 

 1947-48). Ball et al. have now shown that the pn indicator property is characteristic 

 of retinenei in the coupled condition (Ball, Collins, Morton, and Stubbs, 1948). 

 Retinenei condenses spontaneously, as do aldehydes generall}^ with a variety of amino 

 compounds — proteins, amino acids, aromatic amines — and in this state acts as a pn 

 indicator. Indeed a second evidence that the synthetic retinenes added directly or in 

 digitonin solution to our apoenzyme extracts couple with other molecules is that they 

 have acquired this property. They have in fact come to resemble closely the natural 

 products of bleaching rhodopsin and porphjn-opsin in solution. 



A third evidence that synthetic retinene^ couples with other molecules in our 

 enzyme system is that it becomes more and more difficult to extract with fat solvents 

 as the mixture is made more alkaline. If to a solution of retinenci in digitonin one adds 

 methanol in a final concentration of 60 % and shakes vigorously with petroleum ether, 

 almost all the retinene enters the petroleum ether regardless of the pn- But if retinenej 

 in digitonin is mixed with a water extract of the retina prior to carrying out this pro- 

 cedure, smaller and smaller fractions of the retinene enter the petroleum ether as the 

 alkalinity is increased. At pn 4 about 2/3 of the retinene is extracted with petroleum 

 ether in one partition; at pn 9 only about 1/6 of the retinene is extracted. What this 

 probably means is that since retinene^ is coupled by the condensation of its carbonyl 

 group with the amino groups of other molecules, alkalinity favours this process by 

 increasing the proportion of free amino groups, while acidity hinders it by converting 

 amino groups to ammonium ions*. 



The net result of these considerations is that we must regard the normal state of 

 the retinenes in retinas and retinal extracts as a labile equilibrium between free mole- 

 cules and those loosely coupled to other substances. There is no unique retinal molecule, 

 however, with which the retinenes couple and which therefore should be designated 

 "visual yellow" or "indicator yellow". On the contrary, the retinenes regularly condense 

 with a variety of molecules, some protein, some forming fat-soluble complexes. So, for 

 example, when the retinenes have been extracted from retinas with petroleum ether. 



* On observing that retinenej is not readily extracted with petroleum ether from alkaline solu- 

 tions of bleached rhodopsin, Bliss (194S) concluded that it had not been formed. It is formed, but 

 like added retinencj it is retained by coupling with other retinal molecules. 



References p. 228. 



15 



