Spectra Of Pigments Research Articles

Spectroscopic evidence for altered chromophore--protein interactions in low-temperature photoproducts of the visual pigment responsible for congenital night blindness.

The replacement of Gly90 by Asp in human rhodopsin causes congenital night blindness. It has been suggested that the molecular origin for the trait is an altered electrostatic environment of the protonated retinal Schiff base chromophore. We have investigated the corresponding recombinant bovine rhodopsin mutant G90D, as well as the related mutants E113A and G90D/E113A, using spectroscopy at low temperature. This allows the assessment of chromophore-protein interactions under conditions where conformational changes are mainly restricted to the retinal-binding site. Each of the mutant pigments formed bathorhodopsin- and isorhodopsin-like intermediates, but the concomitant visible absorption changes reflected differences in the electrostatic environment of the protonated Schiff base in each pigment. Fourier transform infrared-difference spectroscopy revealed effects on the chromophore fingerprint and hydrogen-out-of-plane vibrational modes, which were indicative of the removal of an electrostatic perturbation near C12 of the retinal chromophore in all three mutants. A comparison of the UV-visible and infrared-difference spectra of the mutant pigments strongly suggests that Glu113 is stably protonated in G90D. The corresponding carbonyl-stretching mode is assigned to a band at 1727 cm-1. In contrast to the case in native bathorhodopsin, the all-trans-retinal chromophores in the primary photoproducts of the mutant pigments are essentially relaxed. The peptide carbonyl vibrational changes in mutants G90D and G90D/ E113A suggest that this is due to a more flexible retinal-binding site. Therefore, the steric strain exerted on the chromophore in native bathorhodopsin may be caused by electrostatic forces that specifically involve glutamate 113.

Analysis of the absorption spectrum of photosystem II reaction centers: temperature dependence, pigment assignment, and inhomogeneous broadening.

In this study a model for decomposition and pigment assignment of the low-temperature (10 K) absorption spectrum of the photosystem II reaction center (D1-D2-cytochrome b559 complex, PSII-RC) is developed. It is based on theoretical calculations of the line shapes of the inhomogeneously broadened pigment spectra, taking into account electron-phonon coupling. The analysis is performed under the hypothesis that exciton coupling is weak, except for the P680 special pair. In this way a detailed decomposition of the absorption spectrum is obtained. Within the model the temperature dependence of the spectrum can be well explained. It is mainly caused by the temperature-dependent changes of the homogeneous absorption spectra of the individual pigments in the PSII-RC. In addition, slight changes in the inhomogeneous distribution functions have to be taken into account. Two slightly different parameter sets are found. We prefer one of these parameter sets which indicates that an accessory chlorophyll (Chl) is the lowest energy pigment in the RC core and that the two antenna Chls have their spectral maxima at 667.7 and 677.9 nm, respectively. The relationship between the shape of the absorption spectrum and the pigment stoichiometry of the sample (ratio of chlorophyll a:pheophytin a), which was noticed by comparison of a variety of different independently prepared samples, can be explained by the presence of "additional" Chl molecules which are nonstoichiometrically bound to part of the PSII-RCs. These Chls can be grouped into three spectrally distinguishable pools. One of them has its absorption maximum at about 683 nm and is responsible for the prominent shoulder that is present in the 10 K absorption spectra of most PSII-RC preparations. Our results suggest that the Chl content of the samples has been underestimated in many spectroscopic studies on the PSII-RC.

Genetic basis of photopigment variations in human dichromats

The spectral sensitivities of the X-encoded pigments in dichromats were studied using the electroretinogram. The action spectra measured for these subjects correspond to four distinctly different X-encoded visual pigments, two different middle-wave pigments spectrally separated by 7 nm and two different long-wave pigments separated by 5 nm. Amino acid sequences were deduced from examination of the genes encoding the pigments. Pairwise comparisons of the opsin structures and pigment spectra confirm and clarify earlier conclusions. Substitutions in exon 5 of the genes produce the spectral difference that separates human X-encoded pigments into middle- and long-wave classes. Polymorphisms in exons 2–4 produce subtypes of pigments that fall within those major classes. Substitution of half of a middle-wave gene with long-wave sequence (exons 1–3) does not shift the middle-wave spectrum. Combined substitutions at positions 230, 233 and 180 produce a 7 nm shift in the middle-wave pigment spectrum. Two subtypes of long-wave pigments that differ in the presence of serine or alanine at position 180 and occur in dichromats, deuteranomalous trichromats, and color normals are spectrally separated by 5–7 run.

Qy-Excitation Resonance Raman Spectra of Bacteriochlorophyll Observed under Fluorescence-Free Conditions. Implications for Cofactor Structure in Photosynthetic Proteins

The importance of obtaining Q{sub y}-execution resonance Raman (RR) spectra of the free photosynthetic pigments promped us to renew the search for conditions under which interference from fluorescence can be mitigated. In this communication, we show that rigorously dried solid films of bacteriochlorophyll (BCh) exhibit sufficiently attenuated emission that Q{sub y}-excitation RR spectra can be acquired. These spectra represent the first Q{sub y} excitation RR spectra of BCh external to a protein matrix. The preliminry results show that certain RR scattering characteristics of BCh in the film are similar to those of the BChs in reaction centers whereas others are very different. The films were prepared by depositing BCh in dry CCl{sub 4} solution onto a quartz window or directly on to the cold tip of a closed-cycle He refrigerator. 15 refs., 1 fig.

Spectroscopic features of native and bleached opio-melanins

Opioid peptides can be converted by tyrosinase into melanin-like compounds, in which the peptide moiety is retained. Such pigments, named opio-melanins, exhibit a characteristic absorption spectrum with a maximum at about 330 nm and a different solubility behaviour with respect to dopa-melanin, being completely soluble in hydrophylic solvents at neutral and basic pH. Opio-melanins precipitate in aqueous solutions below pH 5.0, and show apparent p K a values of 3.1, 3.6 and 4.4 for Tyr-Gly-melanin, Tyr-Gly-Gly-melanin and leuenk-melanin, respectively. The concomitant oxidation of dopa and opioid peptides by tyrosinase produces mixed polymers, showing the distinctive absorption peak at 330 nm. In the dark, in the pH range 5.5–7.0 the pigments are completely stable, whereas H 2O 2 addition provokes a slight degradation. At higher pH values or under simulated solar illumination with or without hydrogen peroxide, bleaching occurs more rapidly than in dopa-melanin. Upon photoirradiation the absorption spectrum of opio-melanins undergoes a marked variation, the peak at 330 nm being replaced by a broad shoulder in the range 280–350 nm. The absorption spectra of native and bleached pigments and the extent of opio-melanins degradation by bleaching agents, confirm the hypothesis that the different initial structure of the precursors accounts for a final diverse polymeric architecture of these pigments with respect to dopa-melanin.

A phycoerythrin‐lacking mutant induced by DCMU in photoheterotrophically grown Nostoc linckia

AbstractA blue‐green strain showing loss of phycoeryhtrin pigment was obtained from the 15 mM fructose and 10−5 M DCMU [3‐(3,4‐dichlorophenyl),1,1‐dimethyl urea] supplemented photoheterotrophic culture of the blackish‐brown rice‐field cyanobacterium Nostoc linckia. Absence of phycoerythrin in the mutant strain was ascertained from the whole cell absorption spectra and spectra of water soluble pigments. Quantitative estimation of photosynthetic pigments of the parent and mutant strain showed that increase in the chlorophyll and phycocyanin content took place in the mutant with a total loss of phycoerythrin. Subculturing under low and high light intensities brought no change in the mutant. The mutant strain grew well, synthesized high amounts of chlorophyll and phycocyanin and possessed higher nitrogen‐fixing capacity in comparison to the parent material.

Polarized site-selected fluorescence spectroscopy of isolated Photosystem I particles

Polarized steady-state fluorescence spectra have been obtained from Photosystem I core complexes of the cyanobacterium Synechocystis PCC 6803 and from LHC-I containing Photosystem I (PS I-200) complexes of spinach by selective laser excitation at 4 K. Excitation above 702 nm in Synechocystis and 720 nm in PS I-200 results in highly polarized emission, suggesting that pigments absorbing at these and longer wavelengths are not able to transfer excitation energy at 4 K. In both systems the peak wavelength of the emission ( λ em) depends strongly on the excitation wavelength ( λ ex). This indicates that in both systems the long-wavelength bands responsible for the steady-state emission are inhomogeneously broadened. The width of the inhomogeneous distribution is estimated to be about 215 cm −1 in Synechocystis and 400 cm −1 in PS I-200. We conclude that the peaks of the total absorption spectra of the long-wavelength pigments of Synechocystis and PS I-200 are at 708 and 716 nm, respectively, and therefore designate these pigments as C-708 and C-716. The results further show that C-708 and C-716 are strongly homogeneously broadened, i.e. carry broad phonon side-bands. The width of these bands is estimated to be about 170 and 200 cm −1 for C-708 and C-716, respectively. The Stokes' shifts appear to be large: about 200 cm -1 (10 nm) for C-708 and about 325 cm −1 (17 nm) for C-716. These values are much higher than usually observed for “normal” antenna pigments, but are in the same order as found previously for a number of dimeric systems. Therefore, we propose that the long-wavelength pigments in Photosystem I are excitonically coupled dimers. Based on fitting with Gaussian bands the presence of one C-708 dimer per P700 is suggested in the core antenna of Synechocystis.

Sequence divergence of the red and green visual pigments in great apes and humans.

We have determined the coding sequences of red and green visual pigment genes of the chimpanzee, gorilla, and orangutan. The deduced amino acid sequences of these pigments are highly homologous to the equivalent human pigments. None of the amino acid differences occurred at sites that were previously shown to influence pigment absorption characteristics. Therefore, we predict the spectra of red and green pigments of the apes to have wavelengths of maximum absorption that differ by < 2 nm from the equivalent human pigments and that color vision in these nonhuman primates will be very similar, if not identical, to that in humans. A total of 14 within-species polymorphisms (6 involving silent substitutions) were observed in the coding sequences of the red and green pigment genes of the great apes. Remarkably, the polymorphisms at 6 of these sites had been observed in human populations, suggesting that they predated the evolution of higher primates. Alleles at polymorphic sites were often shared between the red and green pigment genes. The average synonymous rate of divergence of red from green sequences was approximately 1/10th that estimated for other proteins of higher primates, indicating the involvement of gene conversion in generating these polymorphisms. The high degree of homology and juxtaposition of these two genes on the X chromosome has promoted unequal recombination and/or gene conversion that led to sequence homogenization. However, natural selection operated to maintain the degree of separation in peak absorbance between the red and green pigments that resulted in optimal chromatic discrimination. This represents a unique case of molecular coevolution between two homologous genes that functionally interact at the behavioral level.

Molecular basis for color vision

Amino acid sequences of four kinds of chicken cone pigments and two kinds of nocturnal gecko visual pigment were determined. Calculations of amino acid identities indicate that gecko pigments should be cone pigments. A phylogenetic tree of visual pigments constructed demonstrated that cone pigments evolved earlier than rod pigments (rhodopsins), indicating that daylight vision including color vision appeared earlier than twilight vision. The divergence of cone pigments to rhodopsins would be caused by replacing basic amino acid residues to acidic ones according to net charge calculations. A comparison between chicken rhodopsin and cone pigments (chicken green and red) displayed that the cone pigments are faster in regeneration from 11-cis retinal and opsin, faster in formation of meta II-intermediate and shorter in lifetime of meta II-intermediate than rhodopsin. These facts would partly explain the rapid dark adaptation, the rapid light response and the low photosensitivity of cones compared with rods. In comparison with di- and tri-chromatic color visions, chicken tetra-chromatic vision was discussed on the basis of both absorption spectra of cone pigments and filtering effect of oil droplets.

The ecology of the visual pigments of snappers (Lutjanidae) on the Great Barrier Reef

The visual pigments in the retinal photoreceptors of 12 species of snappers of the genus Lutjanus (Teleostei; Perciformes; Lutjanidae) were measured by microspectrophotometry. All the species were caught on the Great Barrier Reef (Australia) but differ in the colour of the water in which they live. Some live in the clear blue water of the outer reef, some in the greener water of the middle and inshore reefs and some in the more heavily stained mangrove and estuarine water. All the species had double cones, each member of the pair containing a different visual pigment. Using Baker's and Smith's (1982) model to predict the spectral distribution of ambient light from chlorophyll and dissolved organic matter it was found that the absorption spectra of the visual pigments in the double cones were close to those that confer the maximum sensitivity in the different water types. Single cones contained a blue or violet-sensitive visual pigment. The visual pigments in the rods showed little variation, their wavelength of maximum absorption always being in the region 489–502 nm.

R-phycoerythrin from the red alga Gracilaria longa

The red macroalga Gracilaria longa has been investigated for its phycoerythrin content. Absorption and emission spectra of this accessory pigment, purified by gel filtration ( A 565/ A 280≥4.5), showed the typical spectroscopic behaviour of R-phycoerythrin. Absorption maxima were detected at 498, 538 and 565 nm, whereas the emission maximum was observed at 578 nm. The εM (565 nm), calculated for the ‘native’ hexameric phycoerythrin (260 000) was 1.03 × 10 6 M −1 cm −1, each hexamer bearing 25 phycoerythrobilins and nine phycourobilins. The circular dichroism spectrum showed three positive peaks in the region from 495 to 570 nm and two negative bands at 210 and 222 run. The α-helix content, calculated from CD data, was 65%. On SDS-polyacrylamide electrophoresis, the R-phycoerythrin dissociated into four coloured bands (19 000, 21 500, 30 000 and 33 000) corresponding to α, β, γ and γ′ chains. Isoelectric focusing resolved two pairs of major bands, at p I 4.30, 4.35 and p I 4.50, 4.55, and one pair of minor bands at p I 4.70, 4.75.

Light energy conversion with pheophytina monolayer at the SnO2 optically transparent electrode

The photoelectrochemical, absorption and fluorescence properties of pheophytin a mono- and multilayers, deposited on optically transparent tin oxide electrodes and quartz slides were investigated. Spectra of photocurrents coincided with the absorption spectra of photosynthetic pigment in monolayers at the SnO2/solution interfaces. The anodic and cathodic photocurrents were measured at various electrode potentials. Effects of pH, electrode potentials, and concentration of redox reagents on the conversion of solar energy in monolayers on optically transparent electrodes are discussed. The absorption and fluorescence spectral characteristics, and fluorescence lifetime measurements of pheophytina in monolayers and thin films are also discussed in view of the aggregation properties of the photosynthetic pigment. The thermodynamics of adsorption of large amphiphilic compounds at the interface between two immiscible liquids is considered. The adsorption behavior of pheophytin a dissolved in different solvents is investigated. The thermodynamic parameters of pheophytin a adsorption at octane/water and benzene/water interfaces were determined.

Growth and pigmentation in Sphingobacterium antarcticus, a psychrotrophic bacterium from Antarctica

Sphingobacterium antarcticus, a yellow pigmented psychrotrophic bacterium from Antarctica exhibited enhanced pigmentation with increasing temperatures of incubation. This behavior was opposite to mesophilic Sphingobacterium in which pigmentation was reduced upon raising temperatures. The UV-visible spectrum of the crude pigment was characteristic of carotenoids and the pigment gave negative tests for flexirubins. Diphenylamine (DPA), a standard biochemical blocker of carotenoid biosynthesis reduced the growth of broth cultures when grown at extremes of the optimum temperature. Mutants defective in pigmentation were capable of growing between 1–31°C suggesting that pigmentation does not play any role in adapting the bacterium to the psychrotrophic growth temperature. On the other hand, our results suggest that DPA, which is known to block desaturation reactions in the carotenoid biosynthesis pathway may be affecting other desaturation reactions within the bacterium thereby causing reduced growth at extreme temperatures.

ROLE OF HYDROXYL‐BEARING AMINO ACIDS IN DIFFERENTIALLY TUNING THE ABSORPTION SPECTRA OF THE HUMAN RED AND GREEN CONE PIGMENTS

The human red and green cone pigments differ at either 15 or 16 amino acids, depending upon which polymorphic variants are compared. Seven of these amino acid differences involve the introduction or removal of a hydroxyl group. One of these differences, a substitution of alanine for serine at position 180, was found previously to produce a 5 nm blue shift. To determine the role of the remaining six hydroxyl group differences in tuning the absorption spectra of the human red and green pigments, we have studied six site-directed mutants in which single amino acids from the green pigment have been substituted for the corresponding residues in the red pigment. Blue shifts of 7 and 14 nm were observed upon substitution of phenylalanine for tyrosine at position 277 and alanine for threonine at position 285, respectively. Single substitutions at positions 65, 230, 233, and 309 produced spectral shifts of 1 nm or less. These data are in good agreement with a model based upon sequence comparisons among primate pigments and with the properties of site-directed mutants of bovine rhodopsin. Nonadditive effects observed in comparing the absorption spectra of red-green hybrid pigments remain to be explained.

Simple exponential functions describing the absorbance bands of visual pigment spectra

Literature data for visual pigment spectra are formally treated by assuming that the spectra consist of a summation of absorbance bands, that the shape of the bands is invariant according to the Mansfield-MacNichol transform and that this shape is described by simple exponential functions. A new template for constructing visual pigment spectra from peak wavelengths is derived.

In situ characterization of phytoplankton from vertical profiles of fluorescence emission spectra

Vertical profiling of the upper ocean with a laser/fiber optic fluorometer enabled the determination of fluorescence emission spectra of photosynthetic pigments over small vertical scales. Simultaneous acquisition of phycoerythrin (PE) and chlorophyll (chl) emission spectra allowed in situ differentiation between PE-containing cells (cryptomonads and cyanobacteria) and other chl-containing autotrophs. Further, fluorescence spectral peak shifts associated with different species of PE-containing cells resulted in even finer scale in situ taxonomic differentiation. We found that the phycoerythrin fluorescence emission maxima shifted from 578 nm near the surface, to 585 μm at the base of the shallow thermocline (30% light level), and to 590 nm below the thermocline at the base of the euphotic zone (1% light level). These shifts in peak emission coincided with a taxonomic change in the PE-containing cells (as determined from analysis of discrete bottle samples) from a greater proportion of Synechococcus spp. in the upper water column to a greater proportion of cryptomonads at the base of the euphotic zone. These results indicate that the composition of the phytoplankton assemblage may be assessed in situ without sample collection.

ANALYSIS OF WHITE AND BLUE PIGMENTS FROM WATERCOLOURS BY RAMAN MICROSCOPY

Summary The application of Raman microanalysis to the investigation of pigments taken from watercolours is described. The pigment samples identified include calcium carbonate (chalk), titanium dioxide in its anatase crystal form, zinc oxide, barium sulphate and ultramarine taken from a selection of watercolours. The Raman spectra of the white pigments and extenders barium carbonate, lead carbonate, lead sulphate and calcium sulphate (gypsum), and the blue pigments cerulean blue, Prussian blue, indigo, copper phthalocyanin and lapis lazuli, are also reported.

Energy transfer and primary charge separation in Heliobacterium chlorum studied by picosecond time-resolved transient absorption spectroscopy

Transfer of excitation energy to the reaction center and photo-oxidation of the primary electron donor, P798, were studied in membranes of Heliobacterium chlorum with a time-resolution of 10 ps or less. The absorption difference spectrum of excited antenna pigments was dominated by a bleaching around 805 nm and analysis of the kinetics showed that the life-time of excited antenna BChl g was essentially equal (about 20 ps) for all three spectral BChl g species, BChl g 778, BChl g 793 and BChl g 808. This indicates that the excitation energy rapidly equilibrates within the antenna and that most of the excitations have accumulated on BChl g 808 within a few ps after excitation. The 20-ps decay is attributed to energy transfer to the reaction center. After 100 ps, the excited BChl g had dissappeared and the remaining absorption difference signal showed a bleaching maximum at 798 nm, caused by the oxidation of the primary electron donor, P798. The rate of decay of antenna excited states was somewhat faster in the presence of P798 + than with reduced reaction centers, which is in agreement with earlier measurements that showed that P798 + is an efficient quencher of fluorescence (Deinum, G., Kramer, H., Kleinherenbrink, F.A.M., Aartsma, T.J. and Amesz, J. (1991) Biochim. Biophys. Acta 1058, 339–344). The time-resolved absorption difference spectrum also showed a bleaching around 670 nm, due to reduction of the primary electron acceptor, A o, which was induced upon excitation at 585 nm, as well as in the Q y-band of antenna BChl g. From the kinetics at 670 nm, we conclude that the reduction of A o occurs at essentially the same rate as the decay of antenna excitations, indicating that charge-separation is a very rapid process.

Action Spectra for Step‐Up Photophobic Response in Blepharisma

ABSTRACT The cells of Blepharisma which possess red pigment (blepharismin) show step‐up photophobic response (temporal ciliary reversal induced by a sudden increase in light intensity). Bleaching of the cells by cold shock raised a threshold light intensity for the response, Oxidation of red pigment that produced blue pigment did not raise the threshold for the response. The action spectrum for the step‐up photophobic response of the cells which possess normal red pigment had peaks at about 580, 540 and 490 nm, a value which coincided with peaks of an absorption spectrum of the red pigment. The absorption spectrum of oxidized pigment (blue pigment) shifted 20 nm toward infrared light. The action spectrum for the response of the cells which possess blue pigment also shifted 20 nm toward infrared light. Results suggest that red pigment might be involved in the step‐up photophobic response. Key words. Blepharismin, ciliary reversal, photoreceptors, photoresponse.

Rhodopsin levels in the central retinas of normal miniature poodles and those with progressive rod-cone degeneration

Visual pigment in normal miniature poodles and those with progressive rod-cone degeneration (prcd). a late-onset autosomal recessive photoreceptor degeneration, has been studied using imaging fundus reflectometry (IFR). The stage to which the disease had advanced in the animals with prcd was assessed with electroretinography (ERG). Measurements were carried out on seven affected, two heterozygous and three homozygous normal animals. The IFR measurements showed that the in situ difference spectrum of visual pigment measured in the central retina of the normal poodle is typical of vertebrate rhodopsin, with a maximum at about 510 nm. Rhodopsin regeneration following extensive bleaches continues for up to 70 min. In poodles with prcd. rhodopsin is spectrally normal and regenerates at normal rates. In young affected animals under 1 year of age, the final levels of rhodopsin could already be substantially reduced. Serial measurements of visual pigment in these dogs showed differences in the degree and spatial pattern of pigment loss and rate of progression between animals. The extent of visual pigment loss also differed among the older (> 4·5 years) affected animals: while in one animal no pigment could be detected, in another a central band of retina was relatively spared, and significant levels of visual pigment were measured within it. Pigment levels measured within the central 25° of the retinas of poodles heterozygous for prcd were lower than those in normal animals, even though their ERGs were within the normal range.