A sensitive fluorometric technique for the measurement of phycobilin pigments and its application to the study of marine and freshwater picophytoplankton in oligotrophic environments

A sensitive and specific technique is described for the estimation of phycobiliprotein in freshwater and marine picophytoplankton. The method uses fluorescent properties to detect phycoerythrin concentrations as low as 40 ng L-1 from a 1 L water sample and is capable of distinguishing between R-phycoerythrin, C-phycocyanin and C-phycoerythrin. The application of the method to the study of natural picophytoplankton populations in marine and freshwater environments is described. Nitrate concentrations appear to influence picophytoplankton cellular C-phycoerythrin concentrations in surface waters and increasing cellular C-phycoerythrin fluorescence with water depth suggests that this pigment plays a role as a photosynthetic accessory pigment. This revised version was published online in June 2006 with corrections to the Cover Date.     

Chromatic adaptation in lichen phyco- and photobionts

The effect of light quality on the photosynthetic pigments as chromatic adaptation in 8 species of lichens were examined. The chlorophylls, carotenoids in 5 species with green algae as phycobionts (Cladonia mitis, Hypogymnia physodes, H. tubulosa var. tubulosa and subtilis, Flavoparmelia caperata, Xanthoria parietina) and the chlorophyll a, carotenoids and phycobiliprotein pigments in 3 species with cyanobacteria as photobionts (Peltigera canina, P. polydactyla, P. rufescens) were determined. The total content of photosynthetic pigments was calculated according to the formule and particular pigments were determined by means CC, TLC, HPLC and IEC chromatography. The total content of the photosynthetic pigments (chlorophylls, carotenoids) in the thalli was highest in red light (genus Peltigera), yellow light (Xanthoria parietina), green light (Cladonia mitis) and at blue light (Flavoparmelia caperata and both species of Hypogymnia). The biggest content of the biliprotein pigments at red and blue lights was observed. The concentration of C-phycocyanin increased at red light, whereas C-phycoerythrin at green light.     

Effect of light and temperature on the cyanobacterium <Emphasis Type="Italic">Arthronema africanum</Emphasis> - a prospective phycobiliprotein-producing strain

The effect of light intensity (50–300 μmol photons m⁻² s⁻¹) and temperature (15–50°C) on chlorophyll a, carotenoid and phycobiliprotein content in Arthronema africanum biomass was studied. Maximum growth rate was measured at 300 μmol photons m⁻² s⁻¹ and 36°C after 96 h of cultivation. The chlorophyll a content increased along with the increase in light intensity and temperature and reached 2.4% of dry weight at 150 μmol photons m⁻² s⁻¹ and 36°C, but it decreased at higher temperatures. The level of carotenoids did not change significantly under temperature changes at illumination of 50 and 100 μmol photons m⁻² s⁻¹. Carotenoids were about 1% of the dry weight at higher light intensities: 150 and 300 μmol photons m⁻² s⁻¹. Arthronema africanum contained C-phycocyanin and allophycocyanin but no phycoerythrin. The total phycobiliprotein content was extremely high, more than 30% of the dry algal biomass, thus the cyanobacterium could be deemed an alternative producer of C-phycocyanin. A highest total of phycobiliproteins was reached at light intensity of 150 μmol photons m⁻² s⁻¹ and temperature of 36°C, C-phycocyanin and allophycocyanin amounting, respectively, to 23% and 12% of the dry algal biomass. Extremely low (<15°C) and high temperatures (>47°C) decreased phycobiliprotein content regardless of light intensity.     

COMPLEMENTARY CHROMATIC ADAPTATION IN A FILAMENTOUS BLUE-GREEN ALGA

Fluorescent and red light environments generate greatly different patterns of pigmentation and morphology in Fremyella diplosiphon. Most strikingly, red-illuminated cultures contain no measurable C-phycoerythrin and have a mean filament length about 10 times shorter than fluorescent-illuminated cultures. C-phycoerythrin behaves as a photoinducible constituent of this alga. Spectrophotometric and immunochemical procedures were devised so that C-phycoerythrin metabolism could be studied quantitatively with [¹⁴C]-phenylalanine pulse-chased cultures. Transfer of red-illuminated cultures to fluorescent light initiates C-phycoerythrin production by essentially de novo synthesis. C-phycoerythrin is not degraded to any significant extent in cultures continuously illuminated with fluorescent light. Transfer of fluorescent-illuminated cultures to red light causes an abrupt cessation of C-phycoerythrin synthesis. The C-phycoerythrin content of cultures adapting to red light decreases and subsequently becomes constant. Loss of C-phycoerythrin is not brought about by metabolic degradation, but rather by a decrease in mean filament length which is effected by transcellular breakage. In this experimental system, light influences intracellular C-phycoerythrin levels by regulating the rate of synthesis of the chromoprotein.     

Strategy for a protein purification design using C-phycocyanin extract

A variety of techniques have been developed for the separation and recovery of proteins. The cost of purifying the product is frequently determined by the desired quality of the final product, which is evaluated by measuring the purity. In this work the design of a protein purification process for C-phycocyanin, a phycobiliprotein that can be used in the food and medical industries, was established. The study evaluated the use of ammonium sulfate precipitation, ion exchange chromatography and gel filtration to purify C-phycocyanin in a variety of sequences. The final design included the C-phycocyanin extraction step, precipitation with ammonium sulfate and ion exchange chromatography. When the elution step was studied, the kind of elution and pH were considered in order to obtain a product with a final purity of 4.0 with a purification factor of 6.35, so that, at the end of the strategy, C-phycocyanin of analytical grade would be obtained.     

Differentiation between Phycobiliprotein and Colorless Linker Polypeptides by Fluorescence in the Presence of ZnSO(4)

Microcystis aeruginosa, a unicellular cyanobacterium, contains small phycobilisomes consisting of C-phycocyanin, allophycocyanin, and linker polypeptides. SDS-polyacrylamide gels of the phycobilisomes were examined for fluorescent bands before and after spraying with a solution of ZnSO₄, followed by Coomassie brilliant blue staining for protein. This procedure provides a rapid and sensitive method for detecting small amounts of phycobilin-containing polypeptides and distinguishing them from other tetrapyrrole-containing polypeptides and from `colorless' ones. Three polypeptide bands, in addition to the α and β phycobiliprotein subunits, have been detected under these conditions. An 85 kilodalton polypeptide was identified as a phycobiliprotein due to its enhanced fluorescence in the presence of ZnSO₄. The other polypeptides do not contain chromophores and are colorless. They are approximately 34.5 and 30 kilodaltons in size.     

Effect of plastocyanin and phycocyanin on the photosensitivity of chlorophyll-containing bilayer membranes

Summary Photovoltaic effects were studied in bilayer membranes constructed from phosphatidyl choline, monogalactosyl diglyceride, chlorophyll and -carotene. It was demonstrated that the biliprotein C-phycocyanin enhanced the photosensitivity of these membranes. Plastocyanin, an important photosynthetic electron transfer protein, was also found to be effective in enhancing the membrane photovoltage. The C-phycocyanin and plastocyanin were effective on opposite sides of the membrane. Plastocyanin operates to transfer electrons into the membrane, while C-phycocyanin directs electron transfer from the membrane. Membranes containing monogalactosyl diglyceride were found to be extremely stable and were most susceptible to enhancement of photosensitivity by introduction of the proteins. The plastocyanin and C-phycocyanin when used together appeared to operate synergistically.     

Isolation and characterization of phycoerythrocyanin and chromatic adaptation of the thermophilic cyanobacterium Mastigocladus laminosus

The thermophilic cyanobacterium Mastigocladus laminosus exhibits chromatic adaptation: In green light the production of the phycobiliprotein phycoerythrocyanin is enhanced drastically.Phycoerythrocyanin was characterised with respect to its molecular weight, isoelectric point, absorption spectra and size of its aggregates. The two subunits of the protein were separated and characterised according to these criteria. Their chromophore contents, amino acid compositions and N-terminal amino acid sequences were also determined. The sequences were compared with those of allophycocyanin and C-phycocyanin from Mastigocladus laminosus.Abbreviations PEC phycoerythrocyanin - -PEC -subunit of PEC - -PEC -subunit of PEC - PE phycoerythrin - PC phycocyanin - APC allophycocyanin - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - chl a chlorophyll a     

Nitrogen-Fixing Cyanobacterium with a High Phycoerythrin Content

The elemental and molecular composition, pigment content, and productivity of a phycoerythrin-rich nitrogen-fixing cyanobacterium—an Anabaena strain isolated from the coastal lagoon Albufera de Valencia, Spain—has been investigated. When compared with other heterocystous species, this strain exhibits similar chlorophyll a, carotene, and total phycobiliprotein contents but differs remarkably in the relative proportion of specific phycobiliproteins; the content of C-phycoerythrin amounts to 8.3% (versus about 1% in the other species) of cell dry weight. Absorption and fluorescence spectra of intact phycobilisomes isolated from this Anabaena sp. corroborate the marked contribution of phycoerythrin as an antenna pigment, a circumstance that is unusual for cyanobacteria capable of fixing N₂. The pigment content of cells is affected by variations in irradiance and cell density, these adaptive changes being more patent for C-phycoerythrin than for phycocyanins. The Anabaena strain is clumpy and capable of rapid flocculation. It exhibits outdoor productivities higher than 20 g (dry weight) m⁻² day⁻¹ during summer.     

The complete amino-acid sequence of the alpha and beta subunits of B-phycoerythrin from the rhodophytan alga Porphyridium cruentum

Determination of the complete amino-acid sequence of the subunits of B-phycoerythrin from Porphyridium cruentum has shown that the alpha subunit contains 164 amino-acid residues and the beta subunit contains 177 residues. When the sequences of B- and C-phycoerythrins are aligned with those of other phycobiliproteins, it is obvious that B-phycoerythrin lacks a deletion at beta-21-22 present in C-phycoerythrin. However, relative to C-phycoerythrin from Fremyella diplosiphon (Calothrix) (Sidler, W., Kumpf, B., Rüdiger, W. and Zuber, H. (1986) Biol. Chem. Hoppe-Seyler 367, 627-642), B-phycoerythrin has deletions at beta-141k-o, beta-142, beta-143, beta-147 and beta-148. The four singly-linked phycoerythrobilins at positions alpha-84, alpha-143a, beta-84 and beta-155, and the doubly-linked phycoerythrobilin at position beta-50/61 are at sites homologous to the attachment sites in C-phycoerythrin. The aspartyl residues (alpha-87, beta-87, and beta-39), that interact with the bilins at alpha-84, beta-84, and beta-155 in C-phycocyanin, are found in the homologous positions in B-phycoerythrin. B-Phycoerythrin, in common with other phycobiliproteins, contains a N gamma-methylasparagine residue at position beta-72.     

叶绿素缺失和异养生长对蓝藻Synechocystis sp.PCC 6803藻胆蛋白含量的影响

通过遮黑培养缺失ｆｒｘＣ基因的蓝藻Ｓｙｎｅｃｈｏｃｙｓｔｉｓｓｐ．ＰＣＣ６８０３突变工程株,获得了叶绿素缺失的藻细胞,吸收光谱测定及数学计算表明,藻细胞中叶绿素缺失后藻胆蛋白含量增加,藻蓝蛋白和别藻蓝蛋白含量分别为相同条件下野生株对照组的４倍和６倍。野生株遮黑培养时,细胞进行异养生长,藻胆蛋白含量下降,藻蓝蛋白和别藻蓝蛋白含量分别为光照培养条件下自养生长的野生株细胞的３４．５％和２５３％。另外,缺失ａｐｃＥ基因的突变工程株细胞的藻胆蛋白含量也少于对照野生株,表明ａｐｃＥ基因的编码蛋白ＬＣＭ与藻胆蛋白的含量相关。     

Action spectra for nitrate and nitrite assimilation in blue-green algae

Action spectra for the assimilation of nitrate and nitrite have been obtained for several blue-green algae (cyanobacteria) with different accessory pigment composition. The action spectra for both nitrate and nitrite utilization by nitrate-grown Anacystis nidulans L-1402-1 cells exhibited a clear peak at about 620 nanometers, corresponding to photosystem II (PSII) C-phycocyanin absorption, the contribution of chlorophyll a (Chl a) being barely detectable. The action spectrum for nitrate reduction by a nitrite reductase mutant of A. nidulans R2 was very similar. All these action spectra resemble the fluorescence excitation spectrum of cell suspensions of the microalgae monitored at 685 nanometers—the fluorescence band of Chl a in PSII. In contrast, the action spectrum for nitrite utilization by nitrogen-starved A. nidulans cells, which are depleted of C-phycocyanin, showed a maximum near 680 nanometers, attributable to Chl a absorption. The action spectrum for nitrite utilization by Calothrix sp. PCC 7601 cells, which contain both C-phycoerythrin and C-phycocyanin as PSII accessory pigments, presented a plateau in the region from 550 to 630 nanometers. In this case, there was also a clear parallelism between the action spectrum and the fluorescence excitation spectrum, which showed two overlapped peaks with maxima at 562 and 633 nanometers. The correlation observed between the action spectra for both nitrate and nitrite assimilation and the light-harvesting pigment content of the blue-green algae studied strongly suggests that phycobiliproteins perform a direct and active role in these photosynthetic processes.     

葛仙米生长的尺度效应

为考察葛仙米(Nostoc sphaeroides)群体粒径变化对营养成分和光合活性的影响, 测定了生长过程中不同粒径群体的色素成分、光合特性和多糖含量。结果表明: 叶绿素a、藻胆蛋白含量随群体粒径增大而降低; 类胡萝卜素含量在粒径4.85 mm时最高, 藻蓝蛋白/叶绿素a、藻红蛋白/叶绿素a以及别藻蓝蛋白/叶绿素a的比值随粒径增大先降低后升高; 最大潜在光合能力(F_v/F_m)随群体粒径增大逐渐降低, 大群体可通过光系统I介导的环式电子传递链耗散多余能量以保护光合作用系统; 多糖含量在粒径3.03 mm时最高。研究结果显示葛仙米生长具有明显的尺度效应, 细胞组分与光合活性对群体粒径变化具有显著的响应, 开展规模化培养和采收需要关注这一效应。     

Antimelanogenic effect of c-phycocyanin through modulation of tyrosinase expression by upregulation of ERK and downregulation of p38 MAPK signaling pathways

Background  

Pigmentation is one of the essential defense mechanisms against oxidative stress or UV irradiation; however, abnormal hyperpigmentation in human skin may pose a serious aesthetic problem. C-phycocyanin (Cpc) is a phycobiliprotein from spirulina and functions as an antioxidant and a light harvesting protein. Though it is known that spirulina has been used to reduce hyperpigmentation, little literature addresses the antimelanogenic mechanism of Cpc. Herein, we investigated the rationale for the Cpc-induced inhibitory mechanism on melanin synthesis in B16F10 melanoma cells.     

Immunochemistry on cryptomonad biliproteins

A survey is made of the immunochemical behavior of four of the six known types of cryptomonad biliproteins: phycocyanins 612 and 645 and phycoerythrins 545 and 566. They were compared both among themselves and to selected biliproteins isolated from blue-green and red algae. All the cryptomonad biliproteins were shown to be closely related to each other by Ouchterlony double diffusion technics. An antigenic relationship among all the cryptomonad biliproteins and B-phycoerythrin (red alga) and C-phycoerythrin (blue-green alga) was established. Only a very marginal cross-reactivity was found between C-phycocyanin (blue-green algae) and the cryptomonad biliproteins. These results suggest a common ancestor for the photosynthetic units of all three biliprotein-containing phyla.     

叶绿素缺失和异养生长对盐藻Synechocysitis Sp PCC6803藻胆蛋白含量影响

通过遮黑培养缺失frxC基因的蓝藻Synechocystis sp.PCC 6803突变工程株,获得了叶绿素缺失的藻细胞,吸收光谱测定及数学计算表明,藻细胞中叶绿素缺失后藻胆蛋白含量增加,藻蓝蛋白和别藻蓝蛋白含量分别为相同条件下野生株对照组的4倍和6倍。野生株遮黑培养时,细胞进行异养生长, 藻胆蛋白含量下降,藻蓝蛋白和别藻蓝蛋白含量分别为光照培养条件下自养生长的野生株细胞的34.5％和25.3％。另外,缺失apcE基因的突变工程株细胞的藻胆蛋白含量也少于对照野生株,表明apcE基础因的编码蛋白Lcm与藻胆蛋白的含量相关。     

The 1.45 A three-dimensional structure of C-phycocyanin from the thermophilic cyanobacterium Synechococcus elongatus

The conversion of solar radiation to chemical energy by photosynthetic organisms provides the primary driving force for life on earth. Light energy is captured by a variety of pigments, usually bound to proteins, which vary with different types of organisms. We report here the 1.45 A resolution three-dimensional structure of one such pigment protein, C-phycocyanin, from Synechococcus elongatus. The structure is at the highest resolution achieved for any such phycobiliprotein. This level of resolution was made possible by implementing a novel crystallization method whereby nucleation is decoupled from subsequent growth, by incubating crystallizing drops for 7h under nucleation conditions and then transferring them to metastable conditions for growth. This is done without touching the crystallization drops throughout the process.     

Purification and N-terminal analyses of algal biliproteins

1. R-, B- and C-phycoerythrins and R- and C-phycocyanins were isolated and purified on a preparative scale by calcium phosphate chromatography, ammonium sulphate fractionation and crystallization. 2. The N-terminal residues of these biliproteins were analysed. Methionine is the only N-terminal residue of all the phycoerythrins, there being about 14 N-terminal residues per molecule of R- and B-phycoerythrins (mol.wt. 290000) and about 8 per molecule of C-phycoerythrin (mol.wt. 226000). Threonine (1 residue) is N-terminal in C-phycocyanin (mol.wt. 138000), and both threonine (about 1·3 residues) and methionine (5 residues) are N-terminal in R-phycocyanin (mol.wt. 273000). 3. Results suggest that the apoproteins of the various phycoerythrins are closely related, whereas C-phycocyanin has quite a different gross structure, and that R-phycocyanin contains two types of sub-unit, one related to C-phycocyanin and the other to the phycoerythrins.     

Refined three-dimensional structure of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus at 2.7 A

The structure of the phycobiliprotein phycoerythrocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 2.7 A resolution by X-ray diffraction methods on the basis of the molecular model of C-phycocyanin from the same organism. Hexagonal phycoerythrocyanin crystals of space group P6(3) with cell constants a = b = 156.86 A, c = 40.39 A, alpha = beta = 90 degrees, gamma = 120 degrees are almost isomorphous to C-phycocyanin crystals. The crystal structure has been refined by energy-restrained crystallographic refinement and model building. The conventional crystallographic R-factor of the final model was 19.2% with data to 2.7 A resolution. In phycoerythrocyanin, the three (alpha beta)-subunits are arranged around a 3-fold symmetry axis, as in C-phycocyanin. The two structures are very similar. After superposition, the 162 C alpha atoms of the alpha-subunit have a mean difference of 0.71 A and the 171 C alpha atoms of the beta-subunit differ by 0.51 A. The stereochemistry of the chiral atoms in the phycobiliviolin chromophore A84 is C(31)-R, C(4)-S. The configuration of the chromophore is C(10)-Z, C(15)-Z and the conformation C(5)-anti, C(9)-syn and C(14)-anti like the phycocyanobilin chromophores in phycoerythrocyanin and C-phycocyanin.     

Nitrogen-fixing cyanobacteria as source of phycobiliprotein pigments. Composition and growth performance of ten filamentous heterocystous strains

Ten strains of filamentous, heterocystous nitrogen-fixing blue-green algae (cyanobacteria) were screened for growth performance and tolerance to temperature, pH, irradiance and salinity, together with their potential as producers of phycobiliprotein pigments. Phycobiliproteins typically accounted for about 50% total cell protein, the prevalent type being C-phycocyanin, followed by alloppycocyanin, with levels of 17 and 11% d.wt, respectively, in some strains of Anabaena and Nostoc. C-phycoerythrin was the major pigment in several Nostoc strains, reaching 10% d.wt. Some strains represent, therefore, excellent sources of one or more phycobiliproteins. All strains tolerated an irradiance of ca 2000 μmol photon m^-2 s^-1. Anabaena sp. ATCC 33047 and Nostoc sp. (Albufera) exhibited the widest optimum range of both temperature (30–45 and 25–40 °C) and pH (6.5–9.5 and 6.0–9.0) for growth, the former also showing significant salt tolerance. In an outdoor open system, productivity of cultures of two phycoerythrin-rich strains of Nostoc was over 20 g (d.wt) m^-2 d^-1 during summer. The growth performance of the allophycocyanin-rich Anabaena sp. ATCC 33047 in outdoor semi-continuous culture has been assessed throughout the year. Productivity values under optimized conditions ranged from 9 (winter) to 24 (summer) g (d.wt) m^-2 d^-1.