18  Contribution of G. Wald: photochemical sensitivity regulation mechanisms of rods and cones

This hypothesis of Kühne and Hecht must be considered an important insight with regard to biochemical processes underlying dark adaptation. Yet, it only represented a first step towards an understanding of the highly complex processes involved in the photochemical sensitivity regulation mechanisms of rods and cones. Obviously, a deeper understanding would require more information on both the molecular structure of the rod and cone photopigments and the bleaching and regeneration processes generated by light.

18.1  Molecular basis of bleaching

and regeneration of photopigments in rods and cones

Inspired by Hecht, Wald, in the early 1930s, set out to throw light on these largely unexplored research topics (see Wald, 1949a, 1958, 1968). His profound discoveries and insights earned him the Nobel Prize which he shared with Granit and Hartline in 1967.

Firstly, he discovered vitamin A in the retina (Wald, 1933, 1934/1935). Shortly thereafter, he concluded that the photopigment rhodopsin was a conjugated carotenoid-protein engaged in a bleachingregeneration cycle when acted upon by light (Wald, 1934, 1935/1936). Thus, in line with the hypothesis of Kühne and Hecht that light decomposes rhodopsin into its two precursors, Wald presumed that the carotenoids, all-trans retinal (vitamin A aldehyde) and vitamin A represented both photoproducts and precursors of rhodopsin.

A few years later, in 1937, Wald made two new important discoveries:

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1.He showed that in addition to the red-coloured, well-known rhodopsin, there was yet another, purple-coloured rod photopigment which he aptly named porphyropsin (Wald, 1937a).

It turned out that the land vertebrates and marine-fish species possessed the rhodopsin system and freshwater vertebrates, theporphyropsin system, while species which could live both in fresh water and in one of the other habitats frequently possessed both photopigments, mixed or in temporal succession.

As might have been expected on this evidence, the ­chromophores (retinal) of the two rod photopigments were found to be very similar in structure. The principle difference was an extra ­carbon-carbon double bond in the porphyropsin chromophore (situated in the ring chain), displacing the absorption spectrum about 22 nm towards the red-end of the spectrum. It was also found that this change in structure was accompanied by very little change in chemical behaviour; theporphyropsin system constituted a bleaching and regeneration cycle of precisely the same form as that of rhodopsin (see Wald, 1937a, 1938/1939, 1949a, 1968).

2.Wald also discovered the existence of a cone photopigment in the chicken retina (Wald, 1937b). Obviously, the concentration of cone photopigments had to be very low relative to that of rhodopsin, since the outer segment of the cones, in general, appeared quite colourless. Wald, therefore, in his attempt to extract cone photopigments selected the chicken retina which contained mostly cones.

Firstly, he irradiated the retinal extract of the chicken with deep-red light (wavelengths above 650 nm) to which rhodopsin is relatively insensitive, and when the deep-red light produced no further bleaching effect, he exposed the residue to white light and thereby produced a renewed bleaching. The substances which bleached in the red and white light were assumed to be, respectively, cone and rod (rhodopsin) photopigments. The cone photopigment appeared violet in colour (maximum change in spectral absorption due to the red irradiation was obtained at about 570 nm) and Wald therefore named it iodopsin (visual violet).

142 theories of sensitivity regulation

In spite of the predominance of cones in the chicken retina, more rhodopsin than iodopsin was found. In fact, Wald estimated the concentration of iodopsin in single cones to be hundreds of times less than that of rhodopsin in single rods. Thereby, he could explain the relatively low sensitivity of cones and why cones had to be the organ of day vision. Also, he held that the Purkinje shift obtained with the chicken retina could be completely accounted for by a transfer from dependence upon the absorption spectrum of iodopsin in bright light to that of rhodopsin in dim light (see Wald, 1949a).

Wald soon concluded that the cone photopigment iodopsin, like rhodopsin and porphyropsin, was a conjugated carotenoid- ­protein. However, the important question of whether the same ­chromophores represented both the rod and cone systems or whether the ­chromophore of the cone photopigments represented a third variant of retinal remained unanswered.

At last, though, in the 1950s, Wald came up with a sweeping generalization (see Wald, 1958, 1968):

With regard to the molecular structure, all visual photopigments found in the outer segment of vertebrate rods and cones consisted of one of only two types of retinal (the rhodopsin variant retinal1 and theporphyropsin variant retinal2) bound as a chromophore to different proteins called opsins. Wald also recognized two families of opsin, those of the rods and those of the cones, so that, in all, four major photopigments could be synthesized in vertebrate rods and cones. Yet, in order to synthesize the visual pigments the retinal component had to be in a special shape, the 11-cis configuration. This configuration showed both a bend and a twist in the side chain of the retinal molecule which thereby fitted closely to the surface of the opsins.

Along with this parallelism of structure, the different visual photopigments were found to exhibit an extraordinary parallelism of the bleaching-regeneration cycle. We may summarize Wald’s description­ of these two processes as follows:

The only action of light was to isomerize the chromophore of the photopigment from the 11-cis to the all-trans configuration

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(a configuration without the bend and twist, but containing the same chemical constituents). Since the all-trans configuration did not fit into the surface of the opsin like that of the 11-cis retinal, this transformation resulted in significant changes in the molecular structure of the photopigment leading eventually to a complete separation between the all-trans retinal and the opsin. A cascade of change took place. Pre-lumi-rhodopsin, lumi-rhodopsin, meta-rho- dopsin I and meta-rhodopsin II all represented successive, stepwise, and rapid changes in the molecular structure before the compound was hydrolyzed, liberating all-trans retinal. Visual excitation had occurred by the time meta II had been formed (at about 1 ms), since subsequent changes were too slow to be involved.

Finally, the all-trans retinal was converted to vitamin A by an enzymatic process (retinal reductase as apoenzyme and DPN-H2 as coenzyme) in which the retinal molecule received two hydrogen atoms from the coenzyme, reducing its carbonyl group to the primaryalcohol group of vitamin A. Retinal1 was reduced to vitamin A1 and retinal2 to vitamin A2 (see Wald, 1949b). Later, it was found that othercoenzymes may also be used in the enzymatic process (see Wald, 1968).

Up to meta II the all-trans chromophore remained attached toopsin at the same site. As long as this was the case, absorption of a new photon could isomerize the all-trans chromophore to 11-cis retinal and thereby immediately regenerate the photopigment. In fact, the absorption of a second photon by any of the all-trans intermediates of bleaching could re-isomerize the chromophore to 11-cis retinal and thereby regenerate the photopigment. Somewhat surprisingly, then, absorption of light may bleach as well as regenerate photopigments.

Yet, in accord with the hypothesis of Kühne, the photopigments were also found to regenerate after complete bleaching from the vitamin A stage. Thus, retinal was continuously formed in the dark by an enzymatic process in which vitamin A was oxidized by alcohol dehydrogenase (vitamin A1 and A2 were, respectively, oxidized to retinal1 and retinal2). Wald, like Kühne, knew that the pigment