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Partial Analysis of the Bands Seen in Circular Dichroism Spectra of Green and Blue-Green Algal Cells and Thylakoids
Abstract
A comparison was made of the circular dichroism (C.D.) spectra of Chlorella, Euglena, and Anacystis cells and thylakoids. Analyses of the spectra reveal that these C.D. bands are similar to those observed previously in whole spinach choloroplasts and subchloroplast particles. C.D. spectra of Euglena chloroplasts show bands at longer wavelengths than previously reported. From comparisons of circular dichroism spectra and fine structure, it was concluded that: (a) bands seen in circular dichroism spectra were not the result of light scattering from thylakoid membranes; and (b) bands seen in the C.D. spectra of nonmembranous systems (previously reported) could account for circular dichroism of algae. We also concluded that comparisons would have to be made with model systems in order to correct for effects of absorption flattening, concentration obscuring, and differential light scattering of membranous systems.
The quenching of chlorophyll a (Chi a) fluorescence by oxygen was studied using bulk solutions and microdroplets of n-octanol over a Chi a concentration range of 0.1 mu M to 82 mM. Chi a exhibits significant self-quenching (concentration quenching) over this concentration range. The technique of time-resolved fluorescence microscopy was employed to remove the effects of re-absorption. The fluorescence decay curves measured for both aerated and deaerated bulk solutions can be described satisfactorily by a single-exponential function when the concentration of Chi a is not higher than 50 mM. The value of the net rate constant for the fluorescence quenching by oxygen in the bulk solutions (k(q)) shows a distinct dependence on the Chi a concentration for concentrations higher than 1 mM, and reaches a value of (2.0 +/- 0.5) x 10(10) M-1 s(-1) at 25 mM. This value of k(q) is approximately five times larger than that obtained for the most dilute bulk solution. The fluorescence decay curves measured for Chi a in deaerated droplets are approximately exponential, but photodegradation in the aerated droplets is so significant that the fluorescence decays cannot be described by a single-exponential function. Instead, a sum of two exponentials is required, with the longer lived component being attributed to the quenched fluorescence from Chi a, The values of both the quenched and unquenched fluorescence lifetimes decrease with decreasing droplet size, and the net rate constant for the fluorescence quenching by oxygen in the microdroplets (k(q)(D)) is dependent not only on the concentration of Chi a but also on the size of the droplet. The largest value for k(q)(D) of (85 +/- 12) x 10(10) M-1 s(-1) was found in a 4 mu m droplet at a Chi a concentration of 30 mM. This droplet size dependence of the fluorescence quenching by oxygen is ascribed mainly to enhanced energy migration in the smaller droplets, which results in an effective increase in the quenching radius of oxygen.
Analysis of photosynthetic reaction centers from Rhodopseudomonas sphaeroides strains 2.4.1 and Ga shows that each contains approx. 1 mol of a specific carotenoid per mol of reaction center. In strain 2.4.1. the carotenoid is spheroidene (1-methoxy-3,4-didehydro-1,2,7',8',-tetrahydro-psi,psi-carotene); in strain Ga, it is chloroxanthin (1-hydroxy-1, 2, 7', 8'-tetrahydro-psi,psi-carotene). The carotenoid is bound to the same pair of proteins as are the bacteriochlorophylls and bacteriopheophytins of the reaction center. This binding induces strong circular dichroism in the absorption bands of the carotenoid. The carotenoid is close enough to the other pigments of the reaction center so that light energy transfers efficiently from the carotenoid to the bacteriochlorophyll, sensitizing bacteriochlorophyll fluorescence. The fluorescence polarization spectrum of the reaction centers shows that the transition vectors for the visible absorption bands of the carotenoid lie approximately parallel to the 600 nm (Qx) transition of the bacteriochlorophyll complex.
The activity of various electron carriers, including DPIP, spinach plastocyanin, mammalian cytochrome c, and Anabaena cytochrome 553, as donor in the reaction induced by the photochemical system I was examined with lamellar fragments of various algae and spinach.
Reduced DPIP was an effective electron donor irrespective of the organisms, when it was supplied at a high concentration (10⁻³M). Spinach plastocyanin was effective in the reactions with the lamellae of green algae, Euglena, diatom Phaeodactyrum and red algae Porphyra yezoensis and Porphyra sp. Yamamoto II, whereas it was inactive in the lamellae of blue-green algae. Horse-heart cytochrome c and Anabaena cytochrome 553 were active in the reaction with the lamellae of bluegreen algae. The former cytochrome was also active in the reactions in Porphyridium and Cyanidium. The cytochromes were less active in the reactions in which spinach plastocyanin acted as effective electron donor.
The data were interpreted as that the photochemical system I in bluegreen algae differs from that of other photosynthetic organisms with respect to the properties of the site of the electron-input.
Chlorophylls a and b have centres of asymmetry, and possess a degree of circular dichroism (CD) associated with the principal absorption bands (DRATZ et al. 1967, HOUSSIER & SAUER 1970). Dry solutions in solvents such as CC1 contain dimers; these have an increased CD due to exciton splitting (HOUSSIER & SAUER 1970) with a characteristic sigmoid shape. Fragments of thylakoids, prepared by ultrasonic disintegration, show a CD pattern which suggests some dimer content, as well as other unspecified interactions (DRATZ et al. 1967).
Stacking of chloroplast lamellae, isolated from normal and carotenoid mutant chloroplasts of maize (Zea mays L.), was determined after a high-salt treatment.
Stacking of isolated lamellae under favourable ionic conditions was almost identical with that occurring in intact chloroplasts; thus, differences in granum content could be attributed to the architectural properties of lamellae.
Gaussian analyses, performed on the red band of room temperature absorption spectra, have shown that chloroplasts with lamellae of high stacking capacity contain relatively more Chl a662 than chloroplasts containing lamellae of low stacking capacity. The presence of Chl a705–708 was characteristic of preparations containing considerable amounts of stroma lamellae.
This study is a fine structural analysis of the earthworm nervous system and its sheath. The bulk of the findings consists of the addition of fine structural detail to the extant descriptions of the general organization of this system. These findings were of particular interest, (1) that the nervous system possesses one basic type of supportive glial cell, (2) that the zonula occludens was not found anywhere in the structures studied here, and (3) that the sheath of the ventral giant axon is not myelin but consists of a thick covering of many tiny axons and synaptic terminals.
The three-dimensional structure of a chlorophyll-containing protein has
been determined by X-ray crystallography and shown to consist of three
identical subunits, each containing a core of seven bacteriochlorophylls
arranged in an irregular fashion and enclosed within an envelope of
protein.
The molecular exciton model is defined as the resonance interaction between the excited states of loosely bound molecular aggregates (molecular crystals, nonconjugated polymers, molecular laminae, molecular dimers and trimers). The Simpson and Peterson vibrational-electronic coupling cases are applied to spectral characteristics of molecular aggregates. The quasi-classical electrostatic vector model is described for simple molecular aggregates as an approach to understanding the formation of the exciton-state formation. Selection rules and exciton band structures are described for several molecular aggregate structures in the strongcoupling exciton model. In strong-coupling exciton cases, pronounced spectral effects of molecular aggregates compared with individual molecules are predicted, with very fast resonance transfer rates (≲,1015 sec-1) depending on the inverse cube of intermolecular distance. In weak-coupling exciton cases, no conspicuous spectral changes are predicted, but the transfer rates are still high (∼10 12 to 1013 sec-1) and preserve the inverse-cube intermolecular distance dependence. Fö rster's vibrational-relaxation resonance transfer mechanism is emphasized as a contrasting mechanism based on a different theoretical origin (interaction of continua model), with inverse sixth-power dependence on intermolecular distance and much slower transfer rates (106 to 1011 sec-1). Emphasis is placed on the need for new discriminative experimental work. © 2012 by Radiation Research Society. All rights of reproduction in any form reserved.
1.1. An improved procedure has been devised for the purification of large quantities of a detergent-soluble chlorophyll a-protein complex (λmax 677 nm) from a blue-green alga. The complex, which is pure by the criteria of gel electrophoresis and ultracentrifugation, contains approx. 70% of the chlorophyll a of the organism. Studies of the composition of the complex indicate that the protein moiety is probably made up of subunits (mol. wt. 35000), each of which contains five moles of chlorophyll and a trace of β-carotene and echinonone.2.2. Comparison of the physical and chemical properties of the algal chlorophyll-protein complex with those of a water-soluble bacteriochlorophyll-protein of green bacteria, and another detergent-soluble chlorophyll a-protein complex of higher plants suggests that all photosynthetic organisms might contain an analogous class of chlorophyll-proteins (mol. wt. 150000–160000), which are associated with photo-chemical system I activity, and which are composed of four identical protein subunits and 20 moles of chlorophyll.3.3. The water insolubility of the algal and higher plant chlorophyll-protein complexes is most probably explained by the presence of apolar amino acid residues on the surface of the molecule. In vivo the chlorophyll a-proteins are postulated to be attached to the lamellar membrane by hydrophobic bonding of the apolar regions to the lamellar lipids.4.4. Comparative studies indicate that the algal and higher plant complexes are much more closely related to each other than to the bacterial complex.
Treatment of intact cells of two unicellular red algae (Porphyridium spp.) and of an unicellular blue-green alga (Gloeocapsa alpicola) with glutaraldehyde (3% v/v) has only a slight effect on the absorption spectra of their photopigment systems, but stabilizes the normally labile physical association between phycobili-proteins and the chlorophyll-bearing thylacoids. On subsequent breakage of such cells, a substantial fraction of the total phycobiliprotein (30–70%) sediments in a sucrose gradient, in association with the chlorophyll-containing membrane fragments, from which it cannot be dissociated by treatment with detergents.
A study is made of the effect of differential scattering of left and right circularly polarized light on the CD spectra of intact and broken chloroplasts. Dramatic differential light-scattering effects on the spectrum of the intact chloroplasts were observed by varying the fraction of scattered light which is collected by the detector of the CD spectrometer. Evidence is presented which implies that the in-trinsic CD (i.e., after elimination of scattering distortions) of intact chloroplasts is similar to that of nonscattering, sonicated chloroplasts. It is shown that it is not scattering per se, but rather scattering dependent upon the specific ordered arrangement of internal membranes of chloroplasts which produces the large differential scattering effect.
1. The wavelength dependence of the fluorescence polarization (FP) ratio and dichroism has been studied with magneto-oriented (10–13 kG) whole cells of Chlorella pyrenoidosa, Scenedesmus obliquus, Euglena gracilis and spinach chloroplasts suspended in their aqueous growth media (or Tris-buffered sucrose solution in the case of the chloroplasts) under physiological conditions. The FP ratio is defined as the fluorescence intensity polarized parallel divided by the intensity polarized perpendicular to the membrane planes.2. The FP ratio is typically in the range of 1.2–1.9 in Chlorella, 1.20–1.25 in Scenedesmus and 1.4–1.5 in spinach chloroplasts at fluorescence wavelengths above 690 nm. Below 690 nm the FP ratio decreases steadily with decreasing wavelength and may be as low as approx. 1.05 at 660 nm. These results are interpreted in terms of the orientation of the Qy transition moment vectors of the different spectroscopic forms of chlorophyll. For the chlorophyll a 680 form these vectors are inclined at angles of 30° or less (in Chlorella) with respect to the membrane planes, while the shorter wavelength chlorophyll a 670 forms appear to be not nearly as well oriented.3. The Euglena fluorescence peak is red shifted to 714 nm (in the other algae and chloroplasts it is situated at 685 nm) and the FP ratio is approx. 1.20 in the 720–730 nm region and decreases with decreasing wavelength below 720 nm and is only 1.05 at 690 nm. This wavelength dependence is in good qualitative agreement with the fluorescence microscope studies of single chloroplasts of Euglena by Olson, R. A., Butler, W. H. and Jennings, W. H. ((1961) Biochim. Biophys. Acta, 54, 615–617).4. By means of a model calculation it is shown that the high FP ratios observed with Chlorella are entirely consistent with the low values of the degree of polarization (0.01–0.06) determined by previous workers with unoriented cell suspensions.5. The influence of reabsorption and the resulting distortion in the wavelength dependence of the FP ratio are described. The possibility that the fluorescence is polarized by scattering artifacts, rather than being a result of the intrinsic orientation of chlorophyll, is considered.6. Linear dichroism studies with Chlorella and spinach chloroplasts confirm the orientation of the Qy transition moment vectors deduced from the FP ratio. Furthermore, it appears that the porphyrin rings are tilted out of the membrane plane and that the carotenoid molecules tend to lie with their long axes in the lamellar plane.7. In Euglena, dichroism studies indicate that chlorophyll a 680 is unoriented, while chlorophyll a 695 appears to be oriented similar to chlorophyll a 680 in Chlorella or spinach chloroplasts, a result which is also in accord with the measured FP ratio of Euglena.8. The possibility that the magnetic field gives rise to the reorientation of individual chlorophyll molecules is shown to be highly unlikely.
The ability of long-chain unsaturated fatty acids to serve as models for the action of the protein fraction of Ricinus leaf extract on chloroplasts (and algae) has been determined experimentally for several parameters. These include steady-state fluorescence emission and excitation (measured at −196 °), fluorescence induction, light-induced absorption changes of chlorophylls aII and aI, Hill activity, and chloroplast ultrastructure. In addition, the sequential inhibition of system II- and system I-associated electron flow has been demonstrated as a function of increasing concentration of exogenous fatty acid.
Chloroplasts isolated from pea leaves display an intense circular dichroism in the range 600 to 720nm. Circularly polarized light is also differentially scattered by chloroplasts, and this effect can be confused with circular dichroism. By using an instrumental modification it was possible to distinguish, and record separately, the ellipticities of the transmitted light (circular dichroism) and of the scattered light in the same c.d. instrument. By means of a light-scattering apparatus, the intensity of unpolarized light scattered by chloroplasts was measured as a function of wavelength and of angle. This measurement allowed the aforementioned ellipticities to be corrected for mutual interference. At a concentration of 4mug of chlorophyll/ml (the optimum practical concentration of chloroplasts at which there was no significant interaction of scattering and absorption effects) spectra of true circular dichroism (circular differential absorption) and circular differential scattering were obtained. The former showed maxima, positive at 688nm and negative at 676nm, with an intensity Deltatheta=8.3m degrees .litre.(mg of chlorophyll)(-1).cm(-1). The latter had a maximum at 683nm with an intensity of +47m degrees with respect to the solvent baseline; this value is independent of the concentration of chloroplasts in dilute suspensions. It is suggested that the intense circular dichroism of chloroplasts reflects specific chlorophyll-chlorophyll interactions in the light-harvesting pigment. The advantages of this method for determining the c.d. of scattering suspensions over those of other investigators are discussed.
The ability of an exogenous long-chain unsaturated fatty acid (linolenic acid) to induce changes in the circular dichroism (C.D.) spectra of chlorophyllous systems of various levels of organization is demonstrated and attributed to its deaggregating influence. In the case of chlorophyll in solution (CCl(4) or CCl(4)-hexane), deaggregation is by direct action on the chromophore. Evidence is also given for an indirect mechanism when chlorophyll is attached to protein (e.g., in HP-700 complexes); in this case, deaggregation results from a conformational change in the protein. Interpretations are given for the differences in C.D. spectra of nonmembranous and membranous chlorophyll-containing systems. (The latter include "digitonin-isolated" system I particles, subchloroplast particles obtained by means of sonication, and specially prepared intact chloroplasts.)
Chlorophylls a and b undergo aggregation in aqueous-formamide solutions. The chlorophyll a aggregate exhibits absorption bands at 690 mμ and 463 mμ, while that of chlorophyll b are located at 669 mμ and 500 mμ. Studies with pheophytin show that the presence of magnesium is not necessary for aggregation. Less aggregation is observed in the presence of imidazole and urea. The photosensitizing activity and the photoautooxidation of the chlorophyll a aggregate are reported. Chlorophylls a and b are shown to form coaggregates in formamide solutions. Suspensions of formamide-treated chloroplast in buffer undergo spectral shifts similar to those found with hot methanol treatment.
1.1. The effect of a mercurial on circular dichroism (CD) of B-phycoerythrin, R-phycoerythrin and C-phycocyanin has been investigated.2.2. B-phycoerythrin exhibits a double CD band centered at the longest wavelength absorption. Upon treatment with a mercurial, the double CD band appears to reverse its sign resulting in a different double CD band shifted toward shorter wavelengths.3.3. R-phycoerythrin, having three positive CD bands, lacks the double CD band both before and after mercurial treatment.4.4. C-phycocyanin from Anacystis nidulans shows two positive CD bands. The longer wavelength CD band at pH 5 is much greater than the shorter wavelength band. At pH 7, where both bands are of about the same intensity and are well separated, the longer wavelength CD band shifts to a shorter wavelength in the presence of a mercurial.5.5. Subsequent addition of mercaptoethanol brings about a partial regeneration of the longest wavelength positive CD band in the case of both R-phycoerythrin and C-phycocyanin but not with mercurial-treated B-phycoerythrin which produces a double CD band of opposite sign.6.6. These CD changes are discussed in connection with absorption spectral changes as well as with the dissociation and association aspects of these chromoproteins.
SYNOPSIS. In the present study, we show for various photosynthetic organisms in the mature state the diversity which exists in the relative fluorescence intensities of the aggregate and monomer of chlorophyll (at both room temperature and the temperature of liquid nitrogen). For a number of forms, but especially for Euglena gracilis, the spectral transformations which accompany greening are reported. The onset of photosynthetic activity is correlated with a steep (almost step-like) rise in the ratio of aggregate to monomer fluorescence. From this ratio we calculate the “effective” concentration of chlorophyll and estimate lamellar area as a function of greening.
When Euglena gracilis is cultured with light of low intensity (ca. 250 ft-c), an absorption band at 695 mmu is formed in an amount equal to about 20 per cent of the total chlorophyll absorption in this red region. An equally large proportion of C(a)695 is observed in Ochromonas danica, irrespective of light intensity. Other algae tested appear to contain approximately 3 to 5 per cent of their chlorophyll as C(a)695; this proportion does not increase as strikingly with lowering of the light intensity as it does in Euglena. C(a)695 bleaches more readily than the other chlorophyll forms both reversibly, in whole cells, and irreversibly, in homogenates. Cells containing a large proportion of C(a)695 have a fluorescence maximum at 708 mmu, as contrasted to the 687 mmu maximum in other algae. Occasionally, old cultures of Euglena contain cells with an absorption band at approximately 710 mmu. This absorption band is quite stable in aqueous extracts; when the pigment is transferred to ether an equivalent amount of pheophytin a is found to be present. Conditions leading to the formation of the 710 mmu absorption band are not yet known.
Action spectra were obtained for photosystems I and II in chemically fixed algal cells and for photosystem I in unfixed lysozyme treated cells. Untreated algal cells yielded neither of the 2 light reactions with the reaction mixtures used. The action spectra for photosystem I in the blue-green alga Anacystis nidulans and red alga Porphyridium cruentum follow the absorption spectrum of chlorophyll a with a small peak in the region of the accessory pigments. In the green alga Chlorella pyrenoidosa the photosystem I action spectrum follows the absorption spectrum of chlorophyll a. Photosystem II action spectra in A. nidulans and P. cruentum follow the absorption spectra of the accessory pigments while that in C. pyrenoidosa is shifted slightly toward the blue spectral region. These results provide additional evidence that formaldehyde fixed cells are valid models for studying the light reactions of photosynthesis.
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