Isolation, properties and spatial site analysis of γ subunits of B-phycoerythrin and R-phycoerythrin

Polysiphonia urceolata R-phycoerythrin andPorphyridium cruentum B-phycoerythrin were degraded with proteinaseK, and then the nearly native γ subunits were isolated from the reaction mixture. The process of degradation of phycocrythrin with proteinaseK showed that the γ subunit is located in the central cavity of (αβ)₆ hexamer of phycoerythrin. Comparative analysis of the spectra of the native phycoerythrin, the phycoerythrin at pH 12 and the isolated γ subunit showed that the absorption peaks of phycoerythrobilins on α or β subunit are at 535 nm (or 545 nm) and 565 nm, the fluorescence emission maximum at 580 nm; the absorption peak of phycoerythrobilins on the isolated γ subunit is at 589 nm, the fluorescence emission peak at 620 nm which overlaps the absorption maximum of C-phycocyanin and perhaps contributes to the energy transfer with high efficiency between phycoerythrin and phycocyanin in phycobilisome; the absorption maximum of phycourobilin on the isolated γ subunit is at 498 nm, which is the same as that in native phycoerythrin, and the fluorescence emission maximum at 575 nm.     

Fluorescence characteristics of a natural assemblage of freshwater picocyanobacteria

The fluorescence characteristics of a freshwater assemblageof picocyanobacteria weredetermined in a mesotrophic lake usingmicrospectrofluorometry. In Jack's Lake. Ontario. 72–98%of the assemblage was comprised of cells with a single excitationpeak for chlorophyll (Chl) a (emission at 680 nm) at 565 ±3)nm. theexcitation spectra for Chl a resembling the spectralcomposition of downwelling irradiance. The assemblage was, therefore,dominated by a single phycobiliproteinpigment type, similarto type 2 phycoerythrin (PE) marine Synechococcus strains. Theshape of excitation spectra did not change significantly withdepth down to 0.6% of incident irradiance or between samplingdates, although the relative intensity of the PE excitationpeak was generally greater for populations below the thermoclinecompared to surface populations during summer stratification.Two separate populations of PE-containing picocyanobacteria,distinguished based on their morphology and plane of division,could be further separated based on their emission spectra (usingblue excitation): a Synechocystis type cell (PE-Sys) consistentlyhad a more pronounced peak at 665 nm from allophycocyanin comparedto a Synechococcus (PE-Syn) type cell. In addition, the ratioof the PE to Chl a peak emissions was higher in PE-Sys and increasedsignificantly with depth below the thermocline. While nitrogenwas limiting in the lake in summer, experimental additions ofnitrogen did not significantly affect this ratio in surfacewater populations, but increased the ratio in PE-Syn populationsat the base of the photic zone. For surface assemblages of picocyanobacteria,high irradiance may be more-important in regulating fluorescencecharacteristics than nitrogen stress. ¹Deceased September 7, 1993     

One-step chromatography method for efficient separation and purification of R-phycoerythrin from Polysiphonia urceolata

Phycoerythrins have been widely used in food, cosmetics, immunodiagnostics and analytical reagents. An efficient one-step chromatography method for purification of R-phycoerythrins from Polysiphonia urceolata was described in this paper. Pure R-phycoerythrin was obtained with an absorbance ratio A(565)/A(280) of 5.6 and a high recovery yield of 67.33% using a DEAE-Sepharose Fast Flow chromatography with a gradient elution of pH, alternative to common gradient elution of ionic strength. The absorption spectrum of R-phycoerythrin was characterized with three absorbance maxima at 565, 539 and 498 nm, respectively and the fluorescence emission spectrum at room temperature was measured to be 580 nm. The results of native-PAGE, and SDS-PAGE showed no contamination by other proteins in the phycoerythrin solution, which suggests an efficient method for the separation and purification of R-phycoerythrins from Polysiphonia urceolata.     

Characterization of cryptomonad phycoerythrin and phycocyanin

Phycoerythrin, a chromoprotein, from the cryptomonad alga Rhodomonas lens is composed of two pairs of nonidentical polypeptides (α₂β₂). This structure is indicated by a molecular weight of 54,300, calculated from osmotic pressure measurements and by sodium dodecyl sulfate (SDS) gel electrophoresis, which showed bands with molecular weights of 9800 and 17,700 in a 1:1 molar ratio. The s_20,w⁰ of 4.3S is consistent with a protein of this molecular weight. Similar results were obtained with another cryptomonad phycoerythrin and a cryptomonad phycocyanin. Electrophoresis after partial cross-linking by dimethyl suberimidate revealed seven bands for the cryptomonad phycocyanin and six bands for cryptomonad phycoerythrin and confirmed the proposed structure. Spectroscopic studies on α and β subunits of cryptomonad phycocyanin and phycoerythrin were carried out on the separated bands in SDS gels. The individual polypeptides possessed a single absorption band with the following maxima: phycoerythrin (R. lens), α at 565 nm, β at 531 nm; phycocyanin (Chroomonas sp.), α at 644 nm, β at 566 nm. Fluorescence polarization was not constant across the visible absorption band regions of phycoerythrin (R. lens and C. ovata) with higher polarizations located at higher wavelengths, as had also been previously shown for cryptomonad phycocyanin (Chroomonas sp.). Combining the absorption spectra and the polarization results indicates that in each case the β subunit contains sensitizing chromophores and the α subunit fluorescing chromophores. The CD spectra of cryptomonad phycocyanin and both phycoerythrins were similar and were related to the spectra of the individual subunits. In Ouchterlony double-diffusion experiments the cryptomonad phycoerythrins and phycocyanins cross-reacted, with spurring, with phycoerythrin isolated from a red alga. The cryptomonad phycoerythrins were immunochemically very similar to each other and to cryptomonad phycocyanin, with little spurring detected.     

PHOTOCHEMICAL INTERCONVERSION BETWEEN PRECURSORS OF PHYCOBILIN CHROMOPROTEIDS IN TOLYPOTHRIX TENUIS

1. The photochemical conversion between the precursors of phycocyaninand phycoerythrin in Tolypothrix tenuis was investigated.
2. Itwas found that the conversion of phycocyanin-precursor intophycoerythrin-precursor was induced by green light, and thereverse reaction by red light. These reactions proceeded exponentially, indicating that the photochemical process was acceleratedautocatalytically by the reaction-product.
3. The rates of thesephotochemical reactions were found to beunaltered by varyingthe incubation temperature (0? to 35?)and the composition ofthe gas atmosphere (presence or absenceof CO₂ and of O₂ orby an inhibitor of photosynthesis, p-chlorophenyldimethylurea.
4. The action spectra of the photochemical interconversions betweenprecursors of phycobilin chromoproteids were found to be distinctlydifferent from the absorption spectra of chlorophyll and carotenoids.The most effective wavelength for inducing the conversion ofphycocyanin- into phycoerythrin-precursor (541 mµ) isnear the absorption maximum of phycoerythrin (565 mµ),and that of the reverse reaction (641 mµ) is near theabsorption maximum of phycocyanin (620 mµ). Additionaldata, indicating that the phycobilin chromoproteids themselvesdo not participate in these processes as light absorber, werealso presented.
5. On the basis of these results, a possiblemechanism of the photochemicalinterconversion between the precursorsof phycobilin chromoproteidsis proposed.

(Received March 13, 1962; )     

Phycoerythrins of the Red Alga Callithamnion: VARIATION IN PHYCOERYTHROBILIN AND PHYCOUROBILIN CONTENT

Phycoerythrins of several species of the higher red alga Callithamnion show virtually identical spectra, typical of R-phycoerythrins, with absorption maxima at 565, 539, and 497 nanometers. One species, Callithamnion roseum, produces a phycoerythrin lacking the peak at 539 nanometers. Comparison of a “typical” R-phycoerythrin from Callithamnion byssoides with the “atypical” phycoerythrin of C. roseum shows that both proteins carry 35 bilins per native molecule of 240,000 daltons; however, C. byssoides phycoerythrin carries 27.6 phycoerythrobilin and 7.3 phycourobilin groups, whereas C. roseum phycoerythrin carries 24.1 phycoerythrobilin and 10.9 phycourobilin groups. These differences in the relative amounts of the bilin prosthetic groups account in large measure for the differences between the absorption spectra of the native proteins. The ratio of phycoerythrobilin to phycourobilin in C. roseum phycoerythrin can be modulated by varying the light intensity during growth.     

Action spectrum of phototaxis in a cryptomonad alga, Cryptomonas sp.

The action spectrum of the positive topo-phototaxis in Cryptomonaswas determined by photometry in the region of 400 to 680 nmat a stimulus intensity of 8.3 ? 10^–11 Einsteins cm^–2sec^–1. The action spectrum had a main peak at about 560nm unlike peaks for most other flagellated algae. Blue lightwas not very effective and red light above 640 nm had not effect. Swimming rates of individual organisms were measured by darkfield photomicrography. We concluded that the photokinetic effectwas negligible. Among the component pigments of this alga, phycoerythrin hadan absorption spectrum whose main peak appeared to coincidewith that of the phototactic action spectrum. (Received October 31, 1973; )     

Comparison of phycoerythrins (542, 566 nm) from cryptophycean algae

Summary The phycoerythrins from Rhodomonas sp. strain 3-C and Cryptomonas ovata var. palustris were purified and partially characterized. The phycoerythrin from Rhodomonas had a single visible absorption maximum at 542 nm with a shoulder at approximately 562 nm and is, therefore, representative of cryptophyte type I phycoerythrin. The phycoerythrin from C. ovata var. palustris had a single absorption maximum at 566 nm and is, therefore, representative of cryptophyte type III phycoerythrin. Calibrated gel filtration chromatography showed that both of these phycoerythrins have a native molecular weight of 30 800 daltons. Calibrated sodium dodecyl sulfate gel electrophoresis demonstrated that both pigments were composed of two subunits with apparent molecular weights of 17 700 and 11 000 daltons. On polyacrylamide gel electrofocusing both these phycoerythrins had an isoionic point of 4.90.     

Spectral fluorescence signatures in the characterization of phytoplankton community composition

Fluorescence spectral signatures from 28 algal cultures aredescribed.The cultures are split into four groups accordingto their accessory pigments. Phycocyanin and phycoerythrin,characteristic pigments of cyanobacteria, form groups I andII. The characteristic pigment found in group III is chlorophyllb (green and rasinophyte algae) and in group IV it is chlorophyllc (diatoms, dinophytes and some other algae).This preliminarycatalogue of spectral signatures was used to characterize fivenatural phytoplankton communities from brackish water environmentsas a comparison with phytoplankton species found in the samples.Accessory pigments such as phycocyanin and phycoerythrin, characterizinggroups I and II, can be used for identification without confusion.Distinguishing between groups III and IV is more complicated,because their accessory pigments do not have their own fluorescence.These groups can be characterized by increased fluorescenceof chlorophyll a induced by energy excited through chlorophyllb and c. The possibilities of applying the spectral fluorescencesignatures approach to the characterization of natural communitiesare discussed.     

Isolation and characterization of a new phycoerythrin from the cyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. ECS-18

A new phycoerythrin, SCH-phycoerythrin, was purified from Synechococcus sp. ECS-18 by DEAE-Sephacel anion exchange chromatography and Sephacryl S-300 gel filtration. The protein pigment had an absorbance maximum at 542 nm and a fluorescence maximum at 565 nm. The native molecular mass was approximately 219 kDa as determined by gel filtration, and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of two subunits, with molecular mass of 19 and 17.9 kDa. These observations are consistent with the (αβ)₆ subunit composition that is characteristic of phycoerythrins. The α- and β-subunits showed immunological identity by Ouchterlony double immunodiffusion with an anti-phycoerythrin antiserum. The DNA sequence of the SCH-phycoerythrin gene was determined by PCR amplification using primers based on the conserved N-terminal amino acid sequence of the α- and β-subunits of phycoerythrins.     

Some observations on phycobilisomes of Pterocladiella capillacea (Gelidiaceae,Rhodophyta)

ABSTRACT

Phycobilisomes (PBSs) of the red alga Pterocladiella capillacea collected in the field, were characterized both in situ and in vitro by means of a transmission electron microscope (TEM) and an image analyzer. Ultrathin sections of thalli and negatively stained PBSs after isolation revealed hemi-ellipsoidal shapes. In situ PBS dimensions were 38.5 ± 0.2 nm (height) × 38.8 ± 0.2 nm (width) × 22.6 ± 0.2 nm (thickness) in good agreement with the in vitro measurements (mean diameters of 37.6 ± 0.2 nm). These dimensions, especially the width, are smaller than those so far reported for red algae. This could depend either on the ecophysiological conditions of the thalli when harvested and/or on a staggered, symmetrically rotated and compressed disposition of biliprotein rods with respect to the allophycocyanin (APC) core. Hydroxylapatite chromatography of biliproteic extracts and SDS-PAGE electrophoresis revealed that phycoerythrin type R-(λ_max 565 nm>540 nm>498 nm) is formed by α (18.6 kDa), β (19.9 kDa), γ (30.2kDa) and γ′ (33.8 kDa) sub-units. The presence of two γ sub-units suggests that this phycoerythrin is a set of (αβ)₆γ + (αβ)₆γ′ aggregates (R-PE). A spectroscopically distinct form of phycoerythrin with different peak ratios, also found in pure fractions, is thought to be a polydisperse form (the so called r-PE). Similarity of shape and size observed in PBSs both in situ and in vitro, and fluorescence spectral characteristics of PBSs in vitro would indicate a substantial integrity of isolated PBSs. These measurements, if compared with total biliprotein content, would seem to indicate a PE fraction not assembled into PBS. A possible role of phycoerythrin in relation to the ability for a rapid adaptation of this surface species to environmental changes is suggested.     

Action spectra for chromatic adaptation in the blue-green alga Fremyella diplosiphon

Action spectra for chromatic adaptation in Fremyella diplosiphon Drouet have been determined using techniques previously described. Action maxima are at 540 nm, with a half-band width of 80 nm, for induction of phycoerythrin synthesis (green action) and at 650 nm, with a half-band width of 90 nm, for reversal of induction of phycoerythrin synthesis (red action). The red-action spectrum includes a secondary action band centered at ca. 360 nm. Red and green action overlap from 570 to 590 nm with an isosbestic point in the vicinity of 580 nm. Shoulders are present at 520 and 630 nm. Red light is more active than green light. The 540:650-nm quantum effectiveness ratio is 1:7. There is relatively little action of either kind in the blue. The 387:540 nm and 460:650-nm quantum effectiveness ratios are zero. These results contrast strongly with previous determinations in the same organism, with major activity indicated in the blue; they are consistent with the control of photomorphogenesis in the Cyanophyta by a master pigment, analogous to phytochrome.Abbreviations APC allophycocyanin - PC physocyanin - PE phycoerythrin     

Persistence and genetic diversity among strains of phycoerythrinrich cyanobacteria from the picoplankton of Lake Constance

Twelve strains of phycoerythrin (PE)-rich unicellular cyanobacteriaof the Synechococcus type were isolated from the pelagial ofLake Constance in different phases during the growth periodof the year 1994. By analysing the restriction fragment lengthpolymorphism (RFLP) of the DNA three new genotypes were distinguished.The persistence of one strain during the course of the yearwas demonstrated. The data set was compared with the RFLP observedin PE-rich Synechococcus strains isolated in former years fromthe same sampling site. The plurality in the picoplanktic isolatesand the possibility of cultivation of strains is discussed withrespect to the oligotrophication of Lake Constance.     

Action of Near UV and Blue Light on the Photocontrol of Phycobiliprotein Formation; A Complementary Chromatic Adaptation

Action of near UV to blue light on photocontrol of phycoerythrin(PE) and phycocyanin (PC) formation was investigated with non-photobleachedTolypothrix tenuis and Fremyella diplosiphon; this study wasdone to evaluate the proposition of Haury and Bogorad [(1977)Plant Physiol., 60: 835] that near UV to blue light is as effectiveas green and red light for photocontrol of PE and PC formationin blue-green algae and that lack of the blue effect in previousexperiments was due to destruction of blue-absorbing pigment(s)by the photobleaching treatment involved in the experimentalmethod. In our present work, light effect was measured in heterotrophiccultures incubated in darkness following brief exposure to differentwavelengths of light. Results indicated that (1) near UV to blue light was not effectivefor induction of PE formation either in T. tenuis or in F. diplosiphon,and (2) PC formation was induced by near UV light at 360 nmbut not by blue light at 460 nm. These features are identicalwith those previously reported for photobleached cells but notwith those reported by Haury and Bogorad for non-photobleachedcells. We conclude that photobleaching treatment does not haveany influence on the action of near UV to blue light. Actionat 390 and 460 nm observed by Haury and Bogorad probably resultedfrom light effects other than photocontrol, e.g., the actionof photosynthesis. (Received December 18, 1981; Accepted April 8, 1982)     

Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5.

Monoclonal antibodies specific for phycoerythrin (PE) were covalently labeled with the fluorescent dye cyanine 5 (Cy5). Excitation at 488 nm of immune complexes obtained by mixing Cy5-anti-PE with PE resulted in a 4-fold reduction of PE fluorescence measured at 565 nm and an increase of fluorescence measured at 655 nm. The observed energy transfer between PE and Cy5-anti-PE was used to develop three color immunofluorescence staining procedures for flow cytometers equipped with an Argon laser tuned at 488 nm. Mouse IgG1 monoclonal antibodies specific for cell surface antigens were cross-linked with either unlabeled or Cy5 labeled mouse IgG1 anti-PE using F(ab')2 fragments of monoclonal rat anti-mouse IgG1. PE was added to these immune complexes in sufficient amounts to saturate all PE binding sites. Cells were incubated with PE-labeled and PE/Cy5-labeled tetrameric antibody complexes together with FITC labeled antibodies and analyzed by flow cytometry. The emission from FITC, PE and PE/Cy5 could be readily separated and bright three color immunofluorescence staining of mononuclear cells from human peripheral blood and bone marrow was observed. The results of these experiments demonstrate that useful probes for single laser three color staining of cell surface antigens can be readily obtained by mixing of selected reagents. Compared to standard procedures for the covalent labeling of PE (tandem) molecules to antibodies, the non-covalent procedures described in this report provide significant advantages in terms of the amount of reagents, time and equipment required to obtain suitable reagents for three color immunofluorescence staining.     

Effects of Light Quality on Formation of 5-Aminolevulinic Acid, Phycoerythrin and Chlorophyll in Cryptomonas sp. Cells Collected from the Subsurface Chlorophyll Layer

Phycoerythrin obtained from the cells of Cryptomonas sp. (Cryptophyceae)which had been isolated from the subsurface chlorophyll layerin the western Pacific Ocean showed peaks in absorption andfluorescence spectra at 545 and 586 nm, respectively. The rateof photosynthetic O₂ evolution under green light was higherthan those under blue and red light. The rate of 5-aminolevulinic acid (ALA) accumulation in thepresence of levulinic acid was higher under green light thanunder blue and red light. The effects of light quality on therates of O₂ evolution and ALA formation closely resembled eachother. On the other hand, the formation of phycoerythrin andALA was suppressed during growth under blue light. Possible effects of light quality on the formation of photosyntheticpigments in Cryptomonas sp. were discussed. (Received January 31, 1984; Accepted May 14, 1984)     

Wavelength modulation of phycoerythrin synthesis in Synechocystis sp. 6701.

The spectral dependence of phycoerythrin synthesis has been studied in a unicellular photautotrophic cyanobacterium, Synechocystis sp. 6701, in which phycoerythrin synthesis alone is under chromatic control. Cells were partially depleted of their phycobiliprotein pigments through nitrate starvation in the light. Addition of nitrate to the culture medium allowed synthesis of phycobiliproteins in the dark. This synthesis occurred at the expense of the glycogen reserve accumulated during the period of nitrate starvation. Monochromatic irradiations of short duration at lambda less than 590 nm induced increased phycoerythrin synthesis during dark incubation. Monochromatic irradiations of short duration at lambda greater than 590 nm prevented this synthesis. These effects were photoreversible. The spectral distribution showed a maximum at 540 nm for the potentiation of phycoerythrin synthesis, and one at 640 nm for its photoreversal.     

Further evidence for a phycobilisome model from selective dissociation,fluorescence emission,immunoprecipitation, and electron microscopy

Phycobilisomes, isolated in 500 mM Sorensen's phosphate buffer pH 6.8 from the red alga, Porphyridium cruentum, were analyzed by selective dissociation at various phosphate concentrations. The results are consistent with a structural model consisting of an allophycocyanin core, surrounded by a hemispherical layer of R-phycocyanin, with phycoerythrin being on the periphery. Such a structure also allows maximum energy transfer.Intact phycobilisomes transfer excitation energy ultimately to a pigment with a fluorescence emission maximum at 675 nm. This pigment is presumed to be allophycocyanin in an aggregated state. Uncoupling of energy transfer among the pigments, and physical release of the phycobiliproteins from the phycobilisome follow a parallel time-course; phycoerythrin is released first, followed by R-phycocyanin, and then allophycocyanin. In 55 mM phosphate buffer, the times at which 50% of each phycobiliprotein has dissociated are: phycoerythrin 40 min, R-phycocyanin 75 min, and allophycocyanin 140 min.The proposed arrangement of phycobiliproteins within phycobilisomes is also consistent with the results from precipitation reactions with monospecific antisera on intact and dissociated phycobilisomes. Anti-phycoerythrin reacts almost immediately with intact phycobilisomes, but reactivity with anti-R-phycocyanin and anti-allophycocyanin is considerably delayed, suggesting that the antigens are not accessible until a loosening of the phycobilisome structure occurs. Reaction with anti-allophycocyanin is very slow in P. cruentum phycobilisomes, but is much more rapid in phycobilisomes of Nostoc sp. which contains 6–8 times more allophycocyanin. It is proposed that allophycocyanin is partially exposed on the base of isolated intact phycobilisomes of both algae, but that in P. cruentum there are too few accessible sites to permit a rapid formation of a precipitate with anti-allophyocyanin.Phycobilisome dissociation is inversely proportional to phosphate concentration (500 mM to 2 mM), and is essentially unaffected by protein concentration in the range used (30–200 μg/ml). Phycobiliprotein release occurs in the same order (phycoerythrin > R-phycocyanin > allophycocyanin) in the pH range 5.4–8.0.     

Photomorphogenesis in Pteris vittata IV. Action spectra for inhibition of phytochrome-dependent spore germination

Action spectra between 350 and 500 nm for the inhibition ofphytochrome-dependent spore germination in the fern Pteris vittatawere obtained. Both action spectra obtained before and afterred light irradiation have peaks at about 440 nm and 380 nmand shoulders from 440 to 480 nm. These results suggest thatthe phytochrome system is not involved in the inhibitory processof spore germination induced by short irradiation with bluelight. (Received October 8, 1970; )     

Phycoerythrins as chemotaxonomic markers in red algae: A survey

A simple general procedure is described for the purification of high molecular weight phycoerythrin from red algae. Protein of purity adequate for precise spectroscopic characterization was obtained from as little as 0.2 g wet wt of fresh algal tissue. The absorption, excitation and fluorescence emission spectra of over a hundred phycoerythrins from representatives of all of the orders of the Bangiophyceae and Florideophyceae were determined. On the basis of visible absorption spectra, the phycoerythrins were subdivided into five groups: B-phycoerythrin Type I [542 > 567 > 502(s)], B-phycoerythrin Type II [566 ? 528 > 500(s)], R-phycoerythrin Type I (565 > 543 > 497), R-phycoerythrin Type II [566 > 551(s) > 496], and R-phycoerythrin Type III (567 > 539 < 496), where the numbers in parentheses specify the absorption maxima in nm and (s) denotes shoulder. Phycoerythrins do not appear to be useful at familial, ordinal and class levels in taxonomic studies. However, they do appear to be of limited value in discriminating taxonomic groupings at the generic and specific level. Audouinella (Acrochaetiales) can be separated into two groups of species, B-PE and R-PE types, but this is not correlated with cytological, morphological or life-history patterns. Rhodophysema can be removed from the Cryptonemiales and placed in the Acrochaetiales on the basis of its B-PE content, morphology and life history.