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.     

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 (ΔS‡) 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.     

Biochemical aspects of the visual process. XXIII. Sulfhydryl groups and rhodopsin photolysis

1. The number of exposed sulfhydryl groups in cattle rod photoreceptor membranes has been determined in suspension and after solubilization in various detergents both before and after illumination.2. In suspensions, two sulfhydryl groups are modified per mole of rhodopsin, both by Ellman's reagent 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide, while no extra SH groups are uncovered upon illumination. Neither reagent affects the spectral integrity of rhodopsin at 500 nm and the recombination capacity is retained upon modification of both rhodopsin and opsin.3. However, in detergents (digitonin, Triton X-100 and cetyltrimethylammonium bromide (CTAB)) 2–3 additional sulfhydryl groups appear upon illumination, in agreement with earlier reports.4. A total number of six sulfhydryl groups and two disulfide bridges are found in rod photoreceptor membranes, expressed per mole of rhodopsin.5. DTNB reacts somewhat faster with membrane suspensions after than before illumination. The less reactive sulfhydryl modifying agents O-methylisourea and methyl-p-nitrobenzene sulfonate show a similar behavior.6. It is concluded that illumination of rhodopsin in vivo will not uncover additional SH groups, although the reactivity of one exposed SH group may increase somewhat. These findings also exclude a role of SH groups in the covalent binding of the chromophore.     

The rhodopsin system of the squid

Squid rhodopsin (λ_max 493 mµ)—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 (λ_max 500 mµ) near neutrality, yellow (λ_max 380 mµ) in alkaline solution. The two forms—acid and alkaline metarhodopsin—are interconverted according to the equation, Alkaline metarhodopsin + H⁺ acid 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.     

The nature of the gecko visual pigment

Retinal extracts of the Australian gecko, Phyllurus milii (White), have revealed the presence of a photosensitive pigment, unusual for terrestrial animals, because of its absorption maximum at 524 mµ. This pigment has an absorption spectrum which is identical in form with that of other visual chromoproteins. It is not a porphyropsin, for bleaching revealed the presence, not of retinene₂, but of retinene₁ as a chromophore. Photolabile pigments with characteristics similar to those of the Phyllurus visual pigment were also detected in retinal extracts of six other species of nocturnal geckos. The presence of this retinal chromoprotein adequately accounts for the unusual visual sensitivity curve described by Denton for the nocturnal gecko. This pigment may have special biological significance in terms of the unique phylogenetic position of geckos as living representatives of nocturnal animals which retain some of the characteristics of their diurnal ancestors. The occurrence of this retinene₁ pigment, intermediate in spectral position between rhodopsin and iodopsin, is interpreted in support of the transmutation theory of Walls. The results and interpretation of this investigation point up the fact that, from a phylogenetic point of view, too great an emphasis on the duplicity theory may serve to detract attention from the evolutionary history of the retina and the essential unitarianism of the visual cells.     

Calorimetric studies of bovine rod outer segment disk membranes support a monomeric unit for both rhodopsin and opsin

The photoreceptor rhodopsin is a G-protein coupled receptor that has recently been proposed to exist as a dimer or higher order oligomer, in contrast to the previously described monomer, in retinal rod outer segment disk membranes. Rhodopsin exhibits considerably greater thermal stability than opsin (the bleached form of the receptor), which is reflected in an ∼15°C difference in the thermal denaturation temperatures (T_m) of rhodopsin and opsin as measured by differential scanning calorimetry. Here we use differential scanning calorimetry to investigate the effect of partial bleaching of disk membranes on the T_m of rhodopsin and of opsin in native disk membranes, as well as in cross-linked disk membranes in which rhodopsin dimers are known to be present. The T_ms of rhodopsin and opsin are expected to be perturbed if mixed oligomers are present. The T_m remained constant for rhodopsin and opsin in native disks regardless of the level of bleaching. In contrast, the T_m of cross-linked rhodopsin in disk membranes was dependent on the extent of bleaching. The energy of activation for denaturation of rhodopsin and cross-linked rhodopsin was calculated. Cross-linking rhodopsin significantly decreased the energy of activation. We conclude that in native disk membranes, rhodopsin behaves predominantly as a monomer.     

Sulfhydryl groups and the structure of hemoglobin

1. Addition of 2 moles of mersalyl, mercuric chloride, p-chloromercuribenzoate (PCMB), or methyl mercury hydroxide per mole of hemoglobin greatly reduces heme-heme interactions (n), yet these substances have quite different effects on the oxygen affinity (-log p₅₀). Mersalyl and mercuric chloride at this concentration each increase the oxygen affinity, while PCMB and methyl mercury have little or no effect on the oxygen affinity. These effects are primarily associated with the binding of —SH groups, and are largely reversed on the addition of glutathione. —SH groups do not appear to be responsible for the Bohr effect. 2. Evidence is presented for the belief that the two hemes of each half-molecule of horse hemoglobin are situated on either side of a cluster of—SH groups. 3. The mechanism of interaction between the hemes is discussed. It is concluded that the reorganization of the protein architecture which accompanies oxygenation plays a central role in this interaction, in agreement with the views of Pauling and Wyman.     

A new photosensitive pigment of the euryhaline teleost,Gillichthys mirabilis

Retinal extracts have been prepared from dark-adapted mudsuckers by treatment of retinal tissue or of isolated outer segments of the visual cells with digitonin solution. The extracts were examined spectrophotometrically and found to absorb light maximally between the wave lengths of 488 and 510 mµ, depending on the proportion of yellow impurities and light-sensitive pigment present. This photosensitive pigment was shown to be homogeneous by partial bleaching of the extracts with monochromatic light of various wave lengths from 390 to 660 mµ. The mudsucker pigment was specifically demonstrated not to be a mixture of rhodopsin and porphyropsin; the adequacy of the method used to analyze such mixtures was shown by performing a control experiment with an artificial mixture of bullfrog rhodopsin and carp porphyropsin. Comparison of the hydroxylamine difference spectrum and of the absorption maximum of the purest retinal extract located the mudsucker photosensitive pigment maximum at 512 ± 1 mµ. Extraction of retinal tissue with a fat solvent after exposure to white light gave a preparation which after the addition of antimony chloride reagent developed the absorption band maximal near 664 mµ, which is characteristic of retinene₁. If an hour intervened between exposure of the retinal tissue to light and extraction of the carotenoid, the antimony trichloride test gave a color band maximal at 620 mµ, characteristic of vitamin A₁. No evidence of retinene₂ or vitamin A₂ was obtained. The euryhaline mudsucker has, therefore, a photosensitive retinal pigment with an absorption maximum halfway between the peaks of rhodopsins and of porphyropsins and belonging to the retinene₁ system characteristic of rhodopsins. The pigment is therefore named a retinene₁ pigment 512 of the mudsucker, Gillichthys mirabilis. It is uncertain whether this type of photosensitive pigment will be found in other euryhaline fishes.     

The photosensitive retinal pigments of fishes from relatively turbid coastal waters

Digitonin extracts have been prepared from the retinae of a dozen species of marine and euryhaline teleost fishes from turbid water habitats. Spectrophotometric analysis of the extracts shows that the photosensitive retinal pigments of these species have maximum absorption above 500 mµ. In nine species there are retinene₁ pigments with λ_max between 504 and 512 mµ. In the marine but euryhaline mullet, Mugil cephalus, there is a porphyropsin with λ_max 520 mµ. A mixture of rhodopsin and porphyropsin in an extract of a marine puffer, Sphoeroides annulatus, was disclosed by partial bleaching with colored light. In addition, one other species has a 508 mµ pigment, of which the nature of the chromophore was not determined. The habitats in which these fishes live are relatively turbid, with the water greenish or yellowish in color. The spectral transmission of such waters is probably maximal between 520 and 570 mµ. It is suggested that the fishes have become adapted to these conditions by small but significant shifts in spectral absorption of their retinal pigments. These pigments are decidedly more effective than rhodopsin in absorption of wavelengths above 500 mµ. This offers a possible interpretation of the confusing array of retinal pigments described from marine and euryhaline fishes.     

Tautomeric Forms of Metarhodopsin

Light isomerizes the chromophore of rhodopsin, 11-cis retinal (formerly retinene), to the all-trans configuration. This introduces a succession of unstable intermediates—pre-lumirhodopsin, lumirhodopsin, metarhodopsin —in which all-trans retinal is still attached to the chromophoric site on opsin. Finally, retinal is hydrolyzed from opsin. The present experiments show that metarhodopsin exists in two tautomeric forms, metarhodopsins I and II, with λ_max 478 and 380 mµ. Metarhodopsin I appears first, then enters into equilibrium with metarhodopsin II. In this equilibrium, the proportion of metarhodopsin II is favored by higher temperature or pH, neutral salts, and glycerol. The change from metarhodopsin I to II involves the binding of a proton by a group with pK 6.4 (imidazole?), and a large increase of entropy. Metarhodopsin II has been confused earlier with the final mixture of all-trans retinal and opsin (λ_max 387 mµ), which it resembles in spectrum. These two products are, however, readily distinguished experimentally.     

Accessibility of the iodopsin chromophore

Iodopsin can replace its chromophore (11-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH₄ bleaches iodopsin in the dark. In a relatively low concentration of digitonin, the scotopsin (the protein moiety of chicken rhodopsin) removes 11-cis retinal from isopsin in the iodopsin These facts suggests that the linkage of the chromophore to opsin in the iodopsin molecule (presumably a Schiff-base linkage) is accessible to these reagents, which is different from the situation in rhodopsin.     

Immunoreactivity of rhodopsin and opsin

An examination by a radioimmunoassay of the relative affinity of opsin and rhodopsin for rabbit antibody raised against bovine rhodopsin revealed that opsin was the preferred antigen. About 10-fold greater amounts of rhodopsin than opsin were required to achieve 50% inhibition of binding of 125I-labeled ligand in the RIA. Opsin was more reactive when examined in the light or dark, compared to rhodopsin incubated in the dark. Mixtures of opsin and rhodopsin (prepared by partial bleaching of rhodopsin or synthetic mixtures) exhibited increased reactivity with increasing mole fraction of opsin. This response was nonlinear, with small increases in opsin producing relatively large increases in reactivity. A partial fractionation of the antibody into two groups showing differential reactivities toward opsin and rhodopsin was achieved by affinity chromatography on opsin-Sepharose. However, with both groups, opsin was still the preferred antigen. Scatchard analysis of 125I-labeled rhodopsin and opsin produced nonlinear plots, indicating the presence of multiple species of antibody. The affinities and binding capacities were similar for both labeled antigens. In competitive binding studies, the antibody showed a strong preference for either labeled ligand (rhodopsin or opsin) as compared to the unlabeled material. These latter observations indicate that altering rhodopsin either by bleaching or iodination produced changes in the relative immunoreactivity of the molecule.     

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 (λ_max· 500 mµ). Isoretinene a yields a similar light-sensitive pigment, isorhodopsin, the absorption spectrum of which is displaced toward shorter wave lengths (λ_max· 487 mµ). 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 mµ 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 β-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.     

Acid-base properties of rhodopsin and opsin

Purified preparations of cattle rhodopsin have been titrated to various pH, irradiated, and the pH changes followed thereafter until completed. In this way we have obtained the titration curves of rhodopsin, of the immediate product of irradiation, measured within 30 seconds; and of the final product of irradiation (opsin). The rhodopsin preparations display about 54 titratable groups per mole of pigment: about 34 base-binding and 20 acid-binding groups. In default of an absolute purification, one cannot be sure that all of these go with rhodopsin itself. Exposure to light induces an immediate rise of pH between pH 2 and 8, maximal at about pH 5. This—followed by its slow partial or complete reversal—is the only change of pH in the physiological range (6–7). It involves the exposure of 1 new acid-binding group per mole of rhodopsin with pK about 6.6, close therefore to that of the imidazole group of histidine. At acid and alkaline pH this immediate change is followed by slower changes, occupying up to 40 minutes at 20°C. These changes are always in the direction of neutrality. They involve increases of 5 to 6 moles acid bound at acid pH, and 7 moles base bound at alkaline pH. They are associated with the irreversible denaturation of opsin in acid and alkaline solution, as evidenced by loss of its capacity to regenerate rhodopsin. Such frank denaturation procedures as the exposure of rhodopsin to alkali or heat in the dark result in comparable acid-base changes.     

Inherent Instability of the Retinitis Pigmentosa P23H Mutant Opsin

The P23H opsin mutation is the most common cause of autosomal dominant retinitis pigmentosa. Even though the pathobiology of the resulting retinal degeneration has been characterized in several animal models, its complex molecular mechanism is not well understood. Here, we expressed P23H bovine rod opsin in the nervous system of Caenorhabditis elegans. Expression was low due to enhanced protein degradation. The mutant opsin was glycosylated, but the polysaccharide size differed from that of the normal protein. Although P23H opsin aggregated in the nervous system of C. elegans, the pharmacological chaperone 9-cis-retinal stabilized it during biogenesis, producing a variant of rhodopsin called P23H isorhodopsin. In vitro, P23H isorhodopsin folded correctly, formed the appropriate disulfide bond, could be photoactivated but with reduced sensitivity, and underwent Meta II decay at a rate similar to wild type isorhodopsin. In worm neurons, P23H isorhodopsin initiated phototransduction by coupling with the endogenous G_i/o signaling cascade that induced loss of locomotion. Using pharmacological interventions affecting protein synthesis and degradation, we showed that the chromophore could be incorporated either during or after mutant protein translation. However, regeneration of P23H isorhodopsin with chromophore was significantly slower than that of wild type isorhodopsin. This effect, combined with the inherent instability of P23H rhodopsin, could lead to the structural cellular changes and photoreceptor death found in autosomal dominant retinitis pigmentosa. These results also suggest that slow regeneration of P23H rhodopsin could prevent endogenous chromophore-mediated stabilization of rhodopsin in the retina.     

Orientation of Intermediates in the Bleaching of Shear-Oriented Rhodopsin

Cattle rhodopsin can be highly oriented by shearing a wet paste of digitonin micelles of this visual pigment between two quartz slides. This orients the rhodopsin micelles so that their chromophores lie mainly parallel to the direction of shear. In such preparations the orientation of rhodopsin and intermediates of its bleaching by light have been measured with plane-polarized light from -195°C to room temperature. The chromophore maintains essentially the same orientation as in rhodopsin in all the intermediates of bleaching: bathorhodopsin (prelumirhodopsin), lumirhodopsin, and metarhodopsins I and II. When, however, the retinaldehyde chromophore is hydrolyzed from opsin in the presence of hydroxylamine, the retinaldehyde oxime that results rotates so as to lie mainly across the direction of shear. That is, the retinal oxime, though free, orients itself upon the oriented matrix of the opsin-digitonin micelles. These experiments show the rhodopsin-digitonin micelle to be markedly asymmetric, with the chromophore lying parallel to its long axis. The asymmetry could originate in the formation of the micelle, in rhodopsin itself, or by its linear polymerization under the conditions of the experiment. If rhodopsin itself is markedly asymmetric, for which there is some evidence, then, since in the rod outer segments its chromophores lie parallel to the disk membranes, the molecules themselves must lie with their long axes parallel to the membranes.     

Reactions of the Sulfhydryl Groups of Membrane-Bound Bovine Rhodopsin

Reactions of the sulfhydryl groups of bovine rhodopsin in rod outer segment membranes have been investigated using 4,4′-dithiopyridine. This reagent is uncharged at neutral pH and rapidly equilibrates across phospholipid bilayers. Membrane-bound rhodopsin has two kinetically distinguishable sulfhydryl groups reactive to the reagent, this stoichiometry being unchanged by bleaching provided the sulfhydryl reactions themselves are carried out in the dark. The rates of the reactions, however, are substantially increased by bleaching. Irradiation of bleached membranes, either with white light or wavelengths in the neighborhood of 475 nm, results in an increase in the number of reactive sulfhydryls relative to that found for bleached membranes in the dark. A component of the light-driven reaction is dependent on the Ca²⁺ content of the medium.     

Chromcrphore-Environment Interaction of Visual Pigments in Model System

SEVERAL laboratories^1–6 have recently been concerned with the mechanism of the bathochromic shift of about 120 nm which results when 11-cis retinal (λ _max 380 nm) combines with the protein opsin to form rhodopsin (λ_max 498 nm). A red shift of up to 186 nm is involved in the formation of iodopsin from 11-cis retinal and cone opsin^7,8. The active site of bovine rhodopsin consists of the 11-cis retinylidene chromophore attached to a primary amine group of the protein forming a Schiff-base linkage of the type shown in Fig. 1, Ia. On the basis of the chemical reactions of rhodopsin and its derivatives it has been suggested that an interaction between a protonated form of the chromophore (structure of the type Ib) and a lipophilic environment contributes¹¹ to the red shift.     

Studies on cephalopod rhodopsin. Conformational changes in chromophore and protein during the photoregeneration process

The ultraviolet absorbance of squid and octopus rhodopsin changes reversibly at 234 nm and near 280 nm in the interconversion of rhodopsin and metarhodopsin. The absorbance change near 280 nm is ascribed to both protein and chromophore parts. Rhodopsin is photoregenerated from metarhodopsin via an intermediate, P380, on irradiation with yellow light (λ > 520 nm). The ultraviolet absorbance decreases in the change from rhodopsin to metarhodopsin and recovers in two steps; mostly in the process from metarhodopsin to P380 and to a lesser extent in the process from P380 to rhodopsin. P380 has a circular dichroism (CD) band at 380 nm and its magnitude is the same order as that of rhodopsin. Thus it is considered that the molecular structure of P380 is close to that of rhodopsin and that the chromophore is fixed to opsin as in rhodopsin. In the change from metarhodopsin to P380, the chromophore is isomerized from the all-trans to the 11-cis form, and the conformation of opsin changes to fit 11-cis retinal. In the change from P380 to rhodopsin, a small change in the conformation of the protein part and the protonation of the Schiff base, the primary retinal-opsin link, occur.     

Biochemical aspects of the visual process XXVIII. Classification of sulfhydryl groups in rhodopsin and other photoreceptor membrane proteins

Reaction of isolated bovine rod outer segment membrane with radioactiveN-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule.On the basis of their reactivity towardsp-chloromercuribenzoate andp-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with thep-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and is probably located in the vicinity of the chromophoric center.The difference between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) orN-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of thep-chloromercuri-derivatives.