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.     

Single and Multiple Visual Systems in Arthropods

Extraction of two visual pigments from crayfish eyes prompted an electrophysiological examination of the role of visual pigments in the compound eyes of six arthropods. The intact animals were used; in crayfishes isolated eyestalks also. Thresholds were measured in terms of the absolute or relative numbers of photons per flash at various wavelengths needed to evoke a constant amplitude of electroretinogram, usually 50 µv. Two species of crayfish, as well as the green crab, possess blue- and red-sensitive receptors apparently arranged for color discrimination. In the northern crayfish, Orconectes virilis, the spectral sensitivity of the dark-adapted eye is maximal at about 550 mµ, and on adaptation to bright red or blue lights breaks into two functions with λ_max respectively at about 435 and 565 mµ, apparently emanating from different receptors. The swamp crayfish, Procambarus clarkii, displays a maximum sensitivity when dark-adapted at about 570 mµ, that breaks on color adaptation into blue- and red-sensitive functions with λ_max about 450 and 575 mµ, again involving different receptors. Similarly the green crab, Carcinides maenas, presents a dark-adapted sensitivity maximal at about 510 mµ that divides on color adaptation into sensitivity curves maximal near 425 and 565 mµ. Each of these organisms thus possesses an apparatus adequate for at least two-color vision, resembling that of human green-blinds (deuteranopes). The visual pigments of the red-sensitive systems have been extracted from the crayfish eyes. The horse-shoe crab, Limulus, and the lobster each possesses a single visual system, with λ_max respectively at 520 and 525 mµ. Each of these is invariant with color adaptation. In each case the visual pigment had already been identified in extracts. The spider crab, Libinia emarginata, presents another variation. It possesses two visual systems apparently differentiated, not for color discrimination but for use in dim and bright light, like vertebrate rods and cones. The spectral sensitivity of the dark-adapted eye is maximal at about 490 mµ and on light adaptation, whether to blue, red, or white light, is displaced toward shorter wavelengths in what is essentially a reverse Purkinje shift. In all these animals dark adaptation appears to involve two phases: a rapid, hyperbolic fall of log threshold associated probably with visual pigment regeneration, followed by a slow, almost linear fall of log threshold that may be associated with pigment migration.     

Spectral Sensitivity of the Common Prawn, Palaemonetes vulgaris

The vision of Palaemonetes is of particular interest in view of extensive studies of the responses of its chromatophore systems and eye pigments to light. The spectral sensitivity is here examined under conditions of dark adaptation and adaptation to bright colored lights. In each case the relative number of photons per one-fiftieth sec flash needed to evoke a constant peak amplitude (usually 25 or 50 µv) in the electroretinogram (ERG) was measured at various wavelengths throughout the spectrum. The sensitivity is the reciprocal of this number. In dark-adapted animals the spectral sensitivity curve consists of a broad, almost symmetrical band, maximal at about 540 mµ, with a shoulder near 390 mµ. Adaptation to bright red or blue light, left on continuously throughout the measurements, depresses the 540 mµ peak without notably changing its shape or position, implying that only one visual pigment operates in this region. Adaptation to red light, however, spares a violet-sensitive system, so that a high, narrow peak at 390 mµ now dominates the spectral sensitivity function. The 540 and 390 mµ peaks are apparently associated with different visual pigments; and these seem to be segregated in different receptor systems, since the associated ERG's have markedly different time constants. It is suggested that these two sensitivity bands may represent the red- and violet-sensitive components of an apparatus for color differentiation.     

THE RATE OF CO2 ASSIMILATION BY PURPLE BACTERIA AT VARIOUS WAVE LENGTHS OF LIGHT

1. The relative absorption spectrum of the pigments in their natural state in the photosynthetic bacterium Spirillum rubrum is given from 400 to 900 mµ. The position of the absorption maxima in the live bacteria due to each of the pigments is: green pigment, 420, 590, 880; red pigment, 490, 510, 550. 2. The relative absorption spectrum of the green pigment in methyl alcohol has been determined from 400 to 900 mµ. Bands at 410, 605, and 770 mµ were found. 3. The wave length sensitivity curve of the photosynthetic mechanism has been determined and shows maxima at 590 and about 900 mµ. 4. It is concluded that the green bacteriochlorophyll alone and not the red pigment can act as a light absorber for photochemical CO₂ reduction.     

The Spectral Sensitivity of Crayfish and Lobster Vision

(1) The spectral sensitivity function for the compound eye of the crayfish has been determined by recording the retinal action potentials elicited by monochromatic stimuli. Its peak lies at approximately 570 mµ. (2) Similar measurements made on lobster eyes yield functions with maxima in the region of 520 to 525 mµ, which agree well with the absorption spectrum of lobster rhodopsin if minor allowances are made for distortion by known screening pigments. (3) The crayfish sensitivity function, since it is unaffected by selective monochromatic light adaptation, must be determined by a single photosensitive pigment. The absorption maximum of this pigment may be inferred with reasonable accuracy from the sensitivity data. (4) The visual pigment of the crayfish thus has its maximum absorption displaced by 50 to 60 mµ towards the red end of the spectrum from that of the lobster and other marine crustacea. This shift parallels that found in both rod and cone pigments between fresh water and marine vertebrates. In the crayfish, however, an altered protein is responsible for the shift and not a new carotenoid chromophore as in the vertebrates. (5) The existence of this situation in a new group of animals (with photoreceptors which have been evolved independently from those of vertebrates) strengthens the view that there may be strong selection for long wavelength visual sensitivity in fresh water.     

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 EFFECTIVENESS OF THE SPECTRUM IN CHLOROPHYLL FORMATION

1. Although the carotenoid pigments are present in large concentration in the plastids of etiolated Avena seedlings as compared with protochlorophyll, the pigment precursor of chlorophyll, it is possible to show that the carotenoids do not act as filters of the light incident on the plant in the blue region of the spectrum where they absorb heavily. This suggests that the carotenoids are located behind the protochlorophyll molecules in the plastids. 2. Since the carotenoids do not screen and light is necessary for chlorophyll formation, an effectiveness spectrum of protochlorophyll can be obtained which is the reciprocal of the light energy necessary to produce a constant amount of chlorophyll with different wavelengths. The relative effectiveness of sixteen spectral regions in forming chlorophyll was determined. 3. From the effectiveness spectrum, one can conclude that protochlorophyll is a blue-green pigment with major peaks of absorption at 445 mµ, and 645 mµ, and with smaller peaks at 575 and 545 mµ. The blue peak is sharp, narrow, and high, the red peak being broader and shorter. This differs from previous findings where the use of rougher methods indicated that red light was more effective than blue and did not give the position of the peaks of absorption or their relative heights. 4. The protochlorophyll curve is similar to but not identical with chlorophyll. The ratio of the peaks of absorption in the blue as compared to the red is very similar to chlorophyll a, but the position of the peaks resembles chlorophyll b. 5. There is an excellent correspondence between the absorption properties of this "active" protochlorophyll and what is known of the absorption of a chemically known pigment studied in impure extracts of seed coats of the Cucurbitaceae. Conclusive proof of the identity of the two substances awaits chemical purification, but the evidence here favors the view that the pumpkin seed substance, which is chemically chlorophyll a minus two hydrogens, is identical with the precursor of chlorophyll formation found in etiolated plants.     

Electroretinogram Characteristics and the Spectral Mechanisms of the Median Ocellus and the Lateral Eye in Limulus polyphemus

Electrical responses (ERG) to light flashes of various wavelengths and energies were obtained from the dorsal median ocellus and lateral compound eye of Limulus under dark and chromatic light adaptation. Spectral mechanisms were studied by analyzing (a) response waveforms, e.g. response area, rise, and fall times as functions of amplitude, (b) slopes of amplitude-energy functions, and (c) spectral sensitivity functions obtained by the criterion amplitude method. The data for a single spectral mechanism in the lateral eye are (a) response waveforms independent of wavelength, (b) same slope for response-energy functions at all wavelengths, (c) a spectral sensitivity function with a single maximum near 520 mµ, and (d) spectral sensitivity invariance in chromatic adaptation experiments. The data for two spectral mechanisms in the median ocellus are (a) two waveform characteristics depending on wavelength, (b) slopes of response-energy functions steeper for short than for long wavelengths, (c) two spectral sensitivity peaks (360 and 530–535 mµ) when dark-adapted, and (d) selective depression of either spectral sensitivity peak by appropriate chromatic adaptation. The ocellus is 200–320 times more sensitive to UV than to visible light. Both UV and green spectral sensitivity curves agree with Dartnall's nomogram. The hypothesis is favored that the ocellus contains two visual pigments each in a different type of receptor, rather than (a) various absorption bands of a single visual pigment, (b) single visual pigment and a chromatic mask, or (c) fluorescence. With long duration light stimuli a steady-state level followed the transient peak in the ERG from both types of eyes.     

DERIVED PHOTOSENSITIVE PIGMENTS FROM INVERTEBRATE EYES

The red pigment in the eyes of the squid, blue crab, and horseshoe crab becomes photosensitive when treated with formalin, and bleaches in the light. The resulting change in density is approximately symmetrical around a maximum at 480 mµ in the blue green. This difference absorption spectrum is in rough agreement with the spectral sensitivity of the cephalopod eye and differs only slightly from the difference absorption spectrum of vertebrate visual purple. The formalin-sensitized pigment is not melanoid. Its bleaching in squid retinas releases large quantities of retinene. It is suggested that the light sensitivity of the normal squid photopigment may be independent of its light stability.     

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.     

THE PIGMENT-PROTEIN COMPOUND IN PHOTOSYNTHETIC BACTERIA : II. THE ABSORPTION CURVES OF PHOTOSYNTHIN FROM SEVERAL SPECIES OF BACTERIA

Absorption curves have been obtained in the spectral region of 450 to 900 mµ for the water soluble cell juice of four species of photosynthetic bacteria, Spirillum rubrum (strain S1), Rhodovibrio sp. (strain Gaffron), Phaeomonas sp. (strain Delft), and Streptococcus varians (strains C11 and orig.). These curves all show maxima at 790 and 590 mµ due to bacteriochlorophyll, whose highest band, however, occurs at 875, 855, or 840 mµ depending on the species. The bacteria that appear red rather than brown have a band at 550 mµ due to a carotinoid pigment. An absolute absorption curve of bacteriophaeophytin has maxima at 530 and 750 mµ. The extraction of cell juice by supersonic vibration does not change the position of the absorption bands or of the light absorbing capacity of the pigment.     

The Spectral Distribution of Firefly Light. II

The in vivo peak emission wavelengths of bioluminescence are reported for 15 species of American fireflies. A spectrophotometric study of the dorsal light organs of 155 specimens of the Jamaican firefly Pyrophorus plagiophthalamus showed three distinct color distributions with peak emission wavelengths at 550.1 ± 1.5 mµ, 556.8 ± 1.4 mµ, and 562.4 ± 1.0 mµ. Similar spectral measurements of 35 ventral light organs of the same insects gave peak emission wavelengths ranging from 547 through 594 mµ. This is a wider distribution than the total range of all 34 species of firefly studied to date. There was no obvious correlation between the colors of the ventral and dorsal light organs. It appears that P. plagiophthalamus is a special case in which the luciferase enzyme is not only different among members of the same species, but it may be different for the dorsal and ventral light organs in a single individual. A minimum of six different luciferase molecules for P. plagiophthalamus ventral light organs is proposed. The statistical precision in making these spectrophotometric measurements is discussed.     

Structure of the Red Fluorescence Band in Chloroplasts

Using Weber's method of "matrix analysis" for the estimation of the number of fluorescent species contributing to the emission of a sample, it is shown that the fluorescence¹ band in spinach chloroplast fragments at room temperature originates in two species of chlorophyll a. Emission spectra obtained upon excitation with different wavelengths of light (preferentially absorbed in chlorophyll a or b) are presented. Upon cooling to - 196°C, the fluorescence efficiency increases about twentyfold. Two additional bands, that now appear at 696 and 735 mµ, suggest the participation of four molecular species. Emission spectra observed at different concentrations of chloroplast fragments with excitation in chlorophyll a and b and excitation spectra for different concentrations of chloroplast fragments and measurements at 685 and 760 mµ are presented. Two of the four emission bands may belong to pigment system I and two to system II. The 685, 696, and 738 mµ bands respond differently to temperature changes. In the -196°C to -150°C range, the intensity of the 685 mµ band remains constant, and that of the 696 mµ band decreases twice as fast as that of the 738 mµ band.     

Visual Pigments of Goldfish Cones : Spectral Properties and Dichroism

Freshly isolated retinal photoreceptors of goldfish were studied microspectrophotometrically. Absolute absorptance spectra obtained from dark-adapted cone outer segments reaffirm the existence of three spectrally distinct cone types with absorption maxima at 455 ± 3,530 ± 3, and 625 ± 5 nm. These types were found often recognizable by gross cellular morphology. Side-illuminated cone outer segments were dichroic. The measured dichroic ratio for the main absorption band of each type was 2–3:1. Rapidly bleached cells revealed spectral and dichroic transitions in regions near 400–410, 435–455, and 350–360 nm. These photoproducts decay about fivefold as fast as the intermediates in frog rods. The spectral maxima of photoproducts, combined with other evidence, indicate that retinene₂ is the chromophore of all three cone pigments. The average specific optical density for goldfish cone outer segments was found to be 0.0124 ± 0.0015/µm. The spectra of the blue-, and green-absorbing cones appeared to match porphyropsin standards with half-band width Δν = 4,832 ± 100 cm^–1. The red-absorbing spectrum was found narrower, having Δν = 3,625 ± 100 cm^–1. The results are consistent with the notion that visual pigment concentration within the outer segments is about the same for frog rods and goldfish cones, but that the blue-, and green-absorbing pigments possess molar extinctions of 30,000 liter/mol cm. The red-absorbing pigment was found to have extinction of 40,000 liter/mol cm, assuming invariance of oscillator strength among the three cone spectra.     

The two visual pigments of the gecko: The labile behavior

Summary This report describes five selected experiments that describe the labile behavior of pigment-521 of the Tokay gecko and the relatively more stable properties of the second photopigment, pigment-467, of the same retina. Prepared in the chloride-deficient state, P521 is sensitive to mild temperature increases, is destroyed by NH₂OH and NaBH₄ in the dark, responds top-hydroxymercuribenzoate by a spectral shift to shorter wavelengths, exchanges some of its 11 -cis retinal for the 9 -cis isomer in the dark, and reacts to added chloride and nitrate by spectral shifts to longer and shorter wavelengths, respectively. Dissolved in Triton-X-100 it is irreversibly destroyed by only moderate increases in temperature. In all these responses, chloride ions act specifically to protect the pigment. Pigment-467, in contrast, is less sensitive to temperature, is not bleached by NH₂OH and NaBH₄ in the dark, does not exchange its prosthetic group and responds neither to chloride nor to nitrate by the typical P521 effects. With regard to molecular stability and access to the chromophoric structure there appears to be a dual system in the gecko retina with P521 showing similarities to the cone pigment iodopsin; P467 to rhodopsin. It is pointed out that this dual system may be associated with certain responses of the gecko retina that indicate physiological duality. This is the case even though there are no rods and cones, in the classical sense, in the gecko retina.Abbreviations PMB p-hydroxymercuribenzoate - DTT dithiothreitol, Cleland's reagent This work was supported by grant EY-02178 from the National Institutes of Health     

THE PORPHYROPSIN VISUAL SYSTEM

1. In the rods of fresh-water and some anadromous fishes, rhodopsin is replaced by the purple photolabile pigment porphyropsin. This participates in a retinal cycle identical in form with that of rhodopsin, but in which new carotenoids replace retinene and vitamin A. 2. Porphyropsin possesses a broad absorption maximum at 522 ± 2 mµ, and perhaps a minimum at about 430 mµ. The vitamin A-analogue, vitamin A₂, possesses a maximum in chloroform at 355 mµ and yields with antimony trichloride a deep blue color due to a band at 696 mµ. The retinene-analogue, retinene₂, absorbs maximally in chloroform at 405 mµ and possesses an antimony chloride maximum at 706 mµ. 3. Its non-diffusibility through a semi-permeable membrane, salting-out properties, and sensitivity to chemical denaturants and to heat, characterize porphyropsin as a conjugated carotenoid-protein. 4. The porphyropsin cycle may be formulated: porphyropsin See PDF for Structure. retinene₂-protein ₍₂₎ ^→ vitamin A₂-protein ₍₃₎ ^→ porphyropsin. Isolation of the retina cuts this cycle at (3); denaturation procedures or extraction of porphyropsin into aqueous solution eliminate in addition (1) and (2). 5. The primary difference between the rhodopsin and porphyropsin systems appears to be the possession by the latter of an added ethylenic group in the polyene chain.     

The nature of the lamprey visual pigment

From the retina of the land-locked population of the sea lamprey, Petromyzon marinus, a photolabile pigment was extracted which was identified spectrophotometrically as a member of the rhodopsin group of pigments. Using the absorption spectrum of a relatively pure solution and analysis by means of difference spectra, the peak of this pigment was placed at about 497 mµ. The method of selective bleaching by light of different wave lengths revealed no significant amounts of any other pigment in the extracts. A similar pigment was also detected in retinal extracts of the Pacific Coast lamprey, Entospenus tridentatus. These results are significant for two reasons: (a) the lamprey is shown to be an example of an animal which spawns in fresh water but which is characterized by the presence of rhodopsin, rather than porphyropsin, in the retina; (b) the primitive phylogenetic position of the lamprey suggests that rhodopsin was the visual pigment of the original vertebrates.     

PHOTOLYTIC LIPIDS FROM VISUAL PIGMENTS

A method is described for the preservation of iodopsin, the labile photopigment of daylight vision, by freeze drying in vacuo. The lipids released by the action of light on rhodopsin and iodopsin are found to be similar and to possess a labile absorption spectrum in chloroform, with a rising peak at about 390 mµ and a declining peak in the region of 470 mµ. After the change is complete the absorption spectrum resembles closely that of retinene.     

Delayed light action spectra of several algae in visible and ultraviolet light

Action spectra for delayed light production by several algae were determined from 250 to 750 mµ incident light. In the visible portion of the spectrum the action spectra resemble those reported by previous workers for photosynthesis and light emission. Blue-green algae had a maximum at 620 mµ, red algae at 550 mµ, whereas green and brown algae have action spectra corresponding to chlorophyll and carotenoid absorption. In the ultraviolet portion of the spectrum delayed light is emitted by algae down to 250 mµ incident light. The action spectra of the different algae are not alike in the ultraviolet portion of the spectrum. This indicates that pigments other than chlorophyll must be sensitizing or shielding the algae in the ultraviolet region.     

Photosynthetic action spectra of marine algae

A polarographic oxygen determination, with tissue in direct contact with a stationary platinum electrode, has been used to measure the photosynthetic response of marine algae. These were exposed to monochromatic light, of equal energy, at some 35 points through the visible spectrum (derived from a monochromator). Ulva and Monostroma (green algae) show action spectra which correspond very closely to their absorption spectra. Coilodesme (a brown alga) shows almost as good correspondence, including the spectral region absorbed by the carotenoid, fucoxanthin. In green and brown algae, light absorbed by both chlorophyll and carotenoids seems photosynthetically effective, although some inactive absorption by carotenoids is indicated. Action spectra for a wide variety of red algae, however, show marked deviations from their corresponding absorption spectra. The photosynthetic rates are high in the spectral regions absorbed by the water-soluble "phycobilin" pigments (phycoerythrin and phycocyanin), while the light absorbed by chlorophyll and carotenoids is poorly utilized for oxygen production. In red algae containing chiefly phycoerythrin, the action spectrum closely resembles that of the water-extracted pigment, with peaks corresponding to its absorption maxima (495, 540, and 565 mµ). Such algae include Delesseria, Schizymenia, and Porphyrella. In the genus Porphyra, there is a series P. nereocystis, P. naiadum, and P. perforata, with increasingly more phycocyanin and less phycoerythrin: the action spectra reflect this, with increasing activity in the orange-red region (600 to 640 mµ) where phycocyanin absorbs. In all these red algae, photosynthesis is almost minimal at 435 mµ and 675 mµ, where chlorophyll shows maximum absorption. Although the chlorophylls (and carotenoids) are present in quantities comparable to the green algae, their function is apparently not that of a primary light absorber; this role is taken over by the phycobilins. In this respect the red algae (Rhodophyta) appear unique among photosynthetic plants.