Retinaldehyde Research Articles

Monolayer film of rhodopsin at the air/water interface.

MANY reports have been made of chemical and physical properties of rhodopsin1,2. A role of rhodopsin in visual excitation has been fully discussed by Wald3,4. However, most of the work so far has been carried out using aqueous solutions of rhodopsin extracted with digitonin, resulting in a digitonin complex. Accordingly, there is a risk of observing reactions of rhodopsin different from those primarily found in vivo. Recently, electron microscopic findings on the ultra-fine structure of visual cells5,6 suggest that rhodopsin may be located between the double layers of the disk-like units of rod outer-segment which forms a monolayer. It seems useful to pursue the physico-chemical properties of rhodopsin monolayer in vitro in order to postulate models for the molecular function of rhodopsin. Weitzel et al.7 reported properties of the monolayer of vitamin A and its derivatives spread on aqueous solution. Many workers since Devaux8 have investigated the properties of protein films spread on water. We have succeeded in preparing the monolayer film of rhodopsin—vitamin A1 aldehyde combined with protein— at the air/water interface, and have measured the changes of surface pressure following illumination. An extracted rhodopsin solution was dropped on a clean aqueous surface with a micropipette as was the case with spreading a monolayer of protein. The substrate surmounted with rhodopsin consisted of a phosphate buffer solution having pH of 6.8. The surface pressure exerted on the film was measured with a modified method of Sasaki9 under dim red light of wave-length longer than 750 mµ. To plot the film pressure against the area of the spread films, a barrier placed at a distance of 40 cm. from the other fixed barrier was slid on a trough at a constant speed with certain intervals until the distance was reduced to about 25 cm. Thus the plotting of the film pressure yielded a force-area curve. Besides the force-area curve, changes of surface pressure were measured at any area. In the meantime the barrier was kept unslid so that the rhodopsin film might reveal an unstable surface pressure. A rhodopsin solution extracted with diluted ethanol reported by Hosoya et al.10 seemed to be the most suitable agent in preparing a monolayer film. The digitonin extract did not seem to be satisfactory in monolayer experiments, because of the surface activity of digitonin and higher molecular weight of digitonin complex; but it might be observed in comparison with ethanol extract. Fig. 1 shows the force-area curve of the monolayer film of rhodopsin extracted with diluted ethanol. At a low-pressure region, surface pressure increased slowly as the area was diminished.

Vitamin A1 aldehyde in hen's eggs.

WE have recently reported1 that vitamin A1 aldehyde occurs in the ripe eggs of several marine teleost fishes, and is the predominant form of vitamin A in them. In that paper we stated that we could not detect vitamin A1 aldehyde in hen's eggs, but further experiments have shown it to be present.

Studies on the function of vitamin A in mucopolysaccharide biosynthesis

An in vitro system of rat colon segments and of rat colon homogenates was elaborated, which incorporated [ 35S]sulfate and [ 14C]glucose into mucopolysaccharide. Mucopolysaccharide was identified by paper chromatography and paper electrophoresis along with chondroitin sulfate carrier; by co-precipitation and dialysis with chondroitin sulfate; by hydrolysis and identification of labeled sulfate or labeled glucosamine. The homogenate system required adenosine triphosphate, diphosphopyridine nucleotide, glucose and glutamine. The level of incorporation was lowered to about one-half in colon segments and homogenates from vitamin A-deficient rats. Vitamin A, vitamin A-aldehyde and vitamin A-acid (but no other fat-soluble vitamin tested) restored incorporation to normal. With graded levels of vitamin A added, a maximum of incorporation was reached to 10 μg/6 mg of protein in the homogenates. Using glucosamine or galactosamine instead of glucose and glutamine, or uridine diphosphate acetylglucosamine, uridine diphosphate glucuronic acid, uridine diphosphate glucose and acetylglucosamine, incorporation of labeled sulfate was still dependent on vitamin A. This fact shows that the vitamin may be required either in the polymerisation reaction of the uridine derivatives, or the activation or transfer of sulfate. It was shown that the vitamin A-destroying enzyme lipoxidase can lower or abolish incorporation into mucopolysaccharide.

The Effect of Acetone Treatment on Vitamin A1-Aldehyde Extracts from Herring Roe.

The Effect of Acetone Treatment on Vitamin A1-Aldehyde Extracts from Herring Roe.

Vitamin A1 aldehyde in the eggs of the herring (Clupea harengus L.) and other marine teleosts.

Research Article| March 01 1959 Vitamin A1 aldehyde in the eggs of the herring (Clupea harengus L.) and other marine teleosts P. A. Plack; P. A. Plack 1Unit for Biochemical Research Bearing on Fisheries' Problems* at the National Institute for Research in Dairying, University of Reading Search for other works by this author on: This Site PubMed Google Scholar S. K. Kon; S. K. Kon 1Unit for Biochemical Research Bearing on Fisheries' Problems* at the National Institute for Research in Dairying, University of Reading Search for other works by this author on: This Site PubMed Google Scholar S. Y. Thompson S. Y. Thompson 1Unit for Biochemical Research Bearing on Fisheries' Problems* at the National Institute for Research in Dairying, University of Reading Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1959) 71 (3): 467–476. https://doi.org/10.1042/bj0710467 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation P. A. Plack, S. K. Kon, S. Y. Thompson; Vitamin A1 aldehyde in the eggs of the herring (Clupea harengus L.) and other marine teleosts. Biochem J 1 March 1959; 71 (3): 467–476. doi: https://doi.org/10.1042/bj0710467 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1959 CAMBRIDGE UNIVERSITY PRESS1959 Article PDF first page preview Close Modal You do not currently have access to this content.

On the occurrence of vitamin A aldehydes in fish and frog ova

On the occurrence of vitamin A aldehydes in fish and frog ova

The thermal stability of rhodopsin and opsin.

Rhodopsin, the red photosensitive pigment of rod vision, is composed of a specific cis isomer of retinene, neo-b (11-cis), joined as chromophore to a colorless protein, opsin. We have investigated the thermal denaturation of cattle rhodopsin and opsin in aqueous digitonin solution, and in isolated rod outer limbs. Both rhodopsin and opsin are more stable in rods than in solution. In solution as well as in rods, moreover, rhodopsin is considerably more stable than opsin. The chromophore therefore protects opsin against denaturation. This is true whether rhodopsin is extracted from dark-adapted retinas, or synthesized in vitro from neo-b retinene and opsin. Excess neo-b retinene does not protect rhodopsin against denaturation. The protection involves the specific relationship between the chromophore and opsin. Similar, though somewhat less, protection is afforded opsin by the stereoisomeric iso-a (9-cis) chromophore in isorhodopsin. The Arrhenius activation energies (E(a)) and entropies of activation (DeltaSdouble dagger) are much greater for thermal denaturation of rhodopsin and isorhodopsin than of opsin. Furthermore, these values differ considerably for rhodopsins from different species -frog, squid, cattle-presumably due to species differences in the opsins. Heat or light bleaches rhodopsin by different mechanisms, yielding different products. Light stereoisomerizes the retinene chromophore; heat denatures the opsin. Photochemical bleaching therefore yields all-trans retinene and native opsin; thermal bleaching, neo-b retinene and denatured opsin.

Human Rhodopsin

Human rhodopsin in aqueous solution has lambda(max). of 493 mmicro and is lower in the spectrum than the rhodopsins of all other known vertebrates, with the exception of certain deep-sea fishes. Its molar extinction is 40,000 +/- 800. Like other rhodopsins, it bleaches to a mixture of opsin and all-trans retinene and is resynthesized by incubating opsin with neo-b (11-cis) retinene. The regenerated rhodopsin has the same lambda(max). as the extracted pigment; this is due, therefore, not to an unusual retinene but to a characteristic human opsin. The regeneration in solution from opsin and neo-b retinene is a second-order reaction with a half-time, at 29.5 degrees C, of about 2.5 minutes. This is much faster than the synthesis of rhodopsin in the living human eye, and faster than human rod dark-adaptation; the rate of both processes in vivo must be limited by reactions which precede the union of neo-b retinene with opsin, the final step in rhodopsin synthesis. In the rods, rhodopsin is virtually in the solid state-highly oriented in close relation with other highly oriented molecules. In this situation its spectrum is displaced toward the red (lambda(max) 500 mmicro) and is narrower than in solution. For light entering the rods axially, rhodopsin has also a considerably increased extinction, some 1.5 times higher than when randomly oriented. The spectrum of rhodopsin in rods agrees well in form and position with the spectral sensitivity of human rod vision, measured at the retinal surface.

The rhodopsin system of the squid.

Squid rhodopsin (lambda(max) 493 mmicro)-like vertebrate rhodopsins-contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (lambda(max) 500 mmicro) near neutrality, yellow (lambda(max) 380 mmicro) in alkaline solution. The two forms-acid and alkaline metarhodopsin-are interconverted according to the equation, Alkaline metarhodopsin + H(+) right harpoon over left harpoonacid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.

Retinene isomerase.

Rhodopsin is formed by the condensation of opsin with a cis isomer of retinene, called neo-b. The bleaching of rhodopsin releases all-trans retinene which must be isomerized back to neo-b in order for rhodopsin to regenerate. Both retinene isomers are in equilibrium with the corresponding isomers of vitamin A, through the alcohol dehydrogenase system. An enzyme is found in cattle retinas and frog pigment layers which catalyzes the interconversion of all-trans and neo-b retinene. We call it "retinene isomerase." It is soluble in neutral phosphate buffer, and precipitates between 20 and 35 per cent saturation with ammonium sulfate. In the dark, the isomerase converts all-trans and neo-b retinene to an equilibrium mixture of 5 parts neo-b and 95 parts all-trans. With opsin present to trap neo-b, the isomerase catalyzes the synthesis of rhodopsin from all-trans retinene. This reaction, however, is too slow to account for dark adaptation. Retinene is isomerized by light, but too slowly to supply the retina with neo-b. In aqueous solution the pseudoequilibrium mixture contains about 15 per cent neo-b. When all-trans retinene is irradiated in the presence of isomerase, the rate of formation of neo-b is increased about 5 times, and the pseudoequilibrium shifted so that the mixture now contains about 32 per cent neo-b. The isomerase is specific for all-trans and neo-b retinene. It does not act on two other cis isomers of retinene, nor on all-trans or neo-b vitamin A. The role of the isomerase in vision appears to be as follows: in the light, as rhodopsin is bleached to opsin and all-trans retinene, the latter is in part converted to the neo-b isomer and stored in the pigment epithelium as neo-b vitamin A. During dark adaptation, the dominant process is the trapping by opsin of neo-b retinene supplied from stores of neo-b vitamin A, and the slow isomerase-catalyzed "dark" conversion of all-trans to neo-b retinene.

Synthesen in der Carotinoid‐Reihe. 5. Mitteilung. Anwendungen der Reaktion von Whiting

AbstractStarting from various C40‐diols, 7, 7′‐dihydro‐β‐carotene, dehydro‐ retro‐carotene (isocarotene) and three new carotenoids were synthesized by the reaction of Whiting. In addition a new synthesis of β‐carotene from β‐C18‐ketone, acetylene and vitamin A‐aldehyde is described.

Iodopsin.

The iodopsin system found in the cones of the chicken retina is identical with the rhodopsin system in its carotenoids. It differs only in the protein-the opsin -with which carotenoid combines. The cone protein may be called photopsin to distinguish it from the scotopsins of the rods. Iodopsin bleaches in the light to a mixture of photopsin and all-trans retinene. The latter is reduced by alcohol dehydrogenase and cozymase to all-trans vitamin A(1). Iodopsin is resynthesized from photopsin and a cis isomer of vitamin A, neovitamin Ab or the corresponding neoretinene b, the same isomer that forms rhodopsin. The synthesis of iodopsin from photopsin and neoretinene b is a spontaneous reaction. A second cis retinene, isoretinene a, forms iso-iodopsin (lambda(max) 510 mmicro). The bleaching of iodopsin in moderate light is a first-order reaction (Bliss). The synthesis of iodopsin from neoretinene b and opsin is second-order, like that of rhodopsin, but is very much more rapid. At 10 degrees C. the velocity constant for iodopsin synthesis is 527 times that for rhodopsin synthesis. Whereas rhodopsin is reasonably stable in solution from pH 4-9, iodopsin is stable only at pH 5-7, and decays rapidly at more acid or alkaline reactions. The sulfhydryl poison, p-chloromercuribenzoate, blocks the synthesis of iodopsin, as of rhodopsin. It also bleaches iodopsin in concentrations which do not attack rhodopsin. Hydroxylamine also bleaches iodopsin, yet does not poison its synthesis. Hydroxylamine acts by competing with the opsins for retinene. It competes successfully with chicken, cattle, or frog scotopsin, and hence blocks rhodopsin synthesis; but it is less efficient than photopsin in trapping retinene, and hence does not block iodopsin synthesis. Though iodopsin has not yet been prepared in pure form, its absorption spectrum has been computed by two independent procedures. This exhibits an alpha-band with lambda(max) 562 mmicro, a minimum at about 435 mmicro, and a small beta-band in the near ultraviolet at about 370 mmicro. The low concentration of iodopsin in the cones explains to a first approximation their high threshold, and hence their status as organs of daylight vision. The relatively rapid synthesis of iodopsin compared with rhodopsin parallels the relatively rapid dark adaptation of cones compared with rods. A theoretical relation is derived which links the logarithm of the visual sensitivity with the concentration of visual pigment in the rods and cones. Plotted in these terms, the course of rod and cone dark adaptation resembles closely the synthesis of rhodopsin and iodopsin in solution. The spectral sensitivities of rod and cone vision, and hence the Purkinje phenomenon, have their source in the absorption spectra of rhodopsin and iodopsin. In the chicken, for which only rough spectral sensitivity measurements are available, this relation can be demonstrated only approximately. In the pigeon the scotopic sensitivity matches the spectrum of rhodopsin; but the photopic sensitivity is displaced toward the red, largely or wholly through the filtering action of the colored oil globules in the pigeon cones. In cats, guinea pigs, snakes, and frogs, in which no such colored ocular structures intervene, the scotopic and photopic sensitivities match quantitatively the absorption spectra of rhodopsin and iodopsin. In man the scotopic sensitivity matches the absorption spectrum of rhodopsin; but the photopic sensitivity, when not distorted by the yellow pigmentations of the lens and macula lutea, lies at shorter wave lengths than iodopsin. This discrepancy is expected, for the human photopic sensitivity represents a composite of at least three classes of cone concerned with color vision.

The molar extinction of rhodopsin.

The molar extinction of rhodopsin is 40,600 cm.(2) per mole equivalent of retinene; i.e., this is the extinction of a solution of rhodopsin which is produced by, or yields on bleaching, a molar solution of retinene. The molar extinctions of all-trans retinene and all-trans retinene oxime have also been determined in ethyl alcohol and aqueous digitonin solutions. On the assumption that each chromophoric group of rhodopsin is made from a single molecule of retinene, it is concluded that the primary photochemical conversion of rhodopsin to lumi-rhodopsin has a quantum efficiency of 1; though the over-all bleaching of rhodopsin in solution to retinene and opsin may have a quantum efficiency as low as one-half. On bleaching cattle rhodopsin, about two sulfhydryl groups appear for each molecule of retinene liberated. In frog rhodopsin the -SH:retinene ratio appears to be higher, 5:2 or perhaps even 3:1. Some of this sulfhydryl appears to have been engaged in binding retinene to opsin; some may have been exposed as the result of changes in opsin which accompany bleaching, comparable with protein denaturation.

Geometrical isomers of retinene.

Five crystalline retinenes have been isolated, which have every appearance of being cis-trans isomers of one another. They are all-trans retinene; three apparently mono-cis isomers: neoretinenes a and b and isoretinene a; and isoretinene b, an apparently di-cis isomer. The absorption spectra of these substances display the relations expected of cis-trans isomers. The main absorption band is displaced 5.5 to 7 mmicro toward shorter wave lengths for each presumptive cis linkage. Some of the presumptive cis isomers also display a cis peak at 255 to 260 mmicro. All five substances yield an identical blue product on mixing with antimony chloride. All of them are converted by light to what appears to be an identical mixture of stereoisomers. Heat isomerizes them very slowly; only neoretinene b exhibits large changes on heating at 70 degrees C. for 3 hours. The various isomers have been extensively interconverted by gentle procedures, and all of them have been converted to all-trans retinene. The present theory of cis-trans isomerism in carotenoids predicts the existence of four stable isomers of retinene. Instead we seem to have five-specifically three mono-cis forms where two are expected. There is no doubt that all these substances are closely related isomers of one another. The only point in question is whether they differ in part by something other than cis-trans configuration. One possibility, as yet little supported by evidence, is that isomerization between beta- and alpha-ionone rings may be involved. If, however, as seems more likely, all these substances are geometrical isomers of one another, some modification is needed in the present theory of configurational relationships in this class of compounds.

Cis-trans isomers of vitamin A and retinene in the rhodopsin system.

Vitamin A and retinene, the carotenoid precursors of rhodopsin, occur in a variety of molecular shapes, cis-trans isomers of one another. For the synthesis of rhodopsin a specific cis isomer of vitamin A is needed. Ordinary crystalline vitamin A, as also the commercial synthetic product, both primarily all-trans, are ineffective. The main site of isomer specificity is the coupling of retinene with opsin. It is this reaction that requires a specific cis isomer of retinene. The oxidation of vitamin A to retinene by the alcohol dehydrogenase-cozymase system displays only a low degree of isomer specificity. Five isomers of retinene have been isolated in crystalline condition: all-trans; three apparently mono-cis forms, neoretinenes a and b and isoretinene a; and one apparently di-cis isomer, isoretinene b. Neoretinenes a and b were first isolated in our laboratory, and isoretinenes a and b in the Organic Research Laboratory of Distillation Products Industries. Each of these substances is converted to an equilibrium mixture of stereoisomers on simple exposure to light. For this reaction, light is required which retinene can absorb; i.e., blue, violet, or ultraviolet light. Yellow, orange, or red light has little effect. The single geometrical isomers of retinene must therefore be protected from low wave length radiation if their isomerization is to be avoided. By incubation with opsin in the dark, the capacity of each of the retinene isomers to synthesize rhodopsin was examined. All-trans retinene and neoretinene a are inactive. Neoretinene b yields rhodopsin indistinguishable from that extracted from the dark-adapted retina (lambda(max.) 500 mmicro). Isoretinene a yields a similar light-sensitive pigment, isorhodopsin, the absorption spectrum of which is displaced toward shorter wave lengths (lambda(max.) 487 mmicro). Isoretinene b appears to be inactive, but isomerizes preferentially to isoretinene a, which in the presence of opsin is removed to form isorhodopsin before the isomerization can go further. The synthesis of rhodopsin in solution follows the course of a bimolecular reaction, as though one molecule of neoretinene b combines with one of opsin. The synthesis of isorhodopsin displays similar kinetics. The bleaching of rhodopsin, whether by chemical means or by exposure to yellow or orange (i.e., non-isomerizing) light, yields primarily or exclusively all-trans retinene. The same appears to be true of isorhodopsin. The process of bleaching is therefore intrinsically irreversible. The all-trans retinene which results must be isomerized to active configurations before rhodopsin or isorhodopsin can be regenerated. A cycle of isomerization is therefore an integral part of the rhodopsin system. The all-trans retinene which emerges from the bleaching of rhodopsin must be isomerized to neoretinene b before it can go back; or if first reduced to all-trans vitamin A, this must be isomerized to neovitamin Ab before it can regenerate rhodopsin. The retina obtains new supplies of the neo-b isomer: (a) by the isomerization of all-trans retinene in the eye by blue or violet light; (b) by exchanging all-trans vitamin A for new neovitamin Ab from the blood circulation; and (c) the eye tissues may contain enzymes which catalyze the isomerization of retinene and vitamin A in situ. When the all-trans retinene which results from bleaching rhodopsin in orange or yellow light is exposed to blue or violet light, its isomerization is accompanied by a fall in extinction and a shift of absorption spectrum about 5 mmicro toward shorter wave lengths. This is a second photochemical step in the bleaching of rhodopsin. It converts the inactive, all-trans isomer of retinene into a mixture of isomers, from which mixtures of rhodopsin and isorhodopsin can be regenerated. Isorhodopsin, however, is an artefact. There is no evidence that it occurs in the retina; nor has isovitamin Aa or b yet been identified in vivo. In rhodopsin and isorhodopsin, the prosthetic groups appear to retain the cis configurations characteristic of their retinene precursors. In accord with this view, the beta-bands in the absorption spectra of both pigments appear to be cis peaks. The conversion to the all-trans configuration occurs during the process of bleaching. The possibility is discussed that rhodopsin may represent a halochromic complex of a retinyl ion with opsin. The increased resonance associated with the ionic state of retinene might then be responsible both for the color of rhodopsin and for the tendency of retinene to assume the all-trans configuration on its release from the complex. A distinction must be made between the immediate precursor of rhodopsin, neovitamin Ab, and the vitamin A which must be fed in order that rhodopsin be synthesized in vivo. Since vitamin A isomerizes in the body, it is probable that any geometrical isomer can fulfill all the nutritional needs for this vitamin.