发菜藻蓝蛋白分离纯化的研究

以发菜为材料,比较了提取液类型和饱和硫酸铵浓度对藻蓝蛋白提取的影响,并对藻蓝蛋白的提取程序和部分特性进行了研究。结果表明:50 mmol/L KP缓冲液(pH值7.2)是合适的提取液,体积分数为40%~50%饱和硫酸铵盐析效果优于其它浓度。经过DEAE-Toyopeal 650 S离子交换层析和SuperdexTM200凝胶过滤层析后,藻蓝蛋白纯度达6.2,最大吸收峰位于615 nm,荧光发射峰位于649 nm,由α和β2个亚基组成,其分子质量分别为18 051.17和19 142.27 Da。因此,发菜藻蓝蛋白分离纯化较为理想的程序为:藻粉→50 mmol/L KP缓冲液(pH值7.2)浸泡→French pressure(1 500 kg/cm2)破碎细胞→40%~50%饱和硫酸铵盐析→DEAE-Toyopeal 650 S离子交换层析→SuperdexTM200凝胶过滤层析→较纯的藻蓝蛋白。     

Isolation, characterization and antioxidative activity of C-phycocyanin from Limnothrix sp. strain 37-2-1

C-phycocyanin (C-PC) is a blue colored accessory photosynthetic pigment found in cyanobacteria. Some of the medicinal properties of Spirulina have been attributed to this pigment, which includes anticancer, antioxidant, and anti-inflammatory activity. We have screened cyanobacteria isolated from freshwater habitats in Florida for their high content of C-PC. Of 125 strains tested, one filamentous strain identified as Limnothrix sp. was selected for further research. This strain produced 18% C-PC of total dry biomass. Here we describe a simple method for obtaining C-PC of high purity without the use of ion exchange chromatography. The procedure is based on pigment precipitation from the cell lysate with an appropriate concentration of ammonium sulfate, then purification with activated carbon and chitosan, followed by a sample concentration using tangential flow filtration. We have shown that when the lower concentration of ammonium sulfate was used, C-PC with higher purity index was recovered. Characterization of C-PC from Limnothrix showed that it had an absorbance maximum at 620nm and fluorescence at 639nm. The molecular mass of intact C-PC was estimated to be ~50kDa with α and β subunits forming dimmers. When C-PC content per unit biomass was compared to that of marketed Spirulina powder, we found that Limnothrix was superior. C-phycocyanin from Limnothrix had an antioxidative activity on DPPH free radicals similar to that found in a natural antioxidant - rutin.     

Deconvolution of C-phycocyanin beta-84 and beta-155 chromophore absorption and fluorescence spectra of cyanobacterium Mastigocladus laminosus.

Absorption and fluorescence spectra of the C-phycocyanin beta-subunit were quantitatively deconvoluted into component spectra of the beta-84 and beta-155 chromophores. The deconvolution procedure was based on a theoretical treatment of polarization properties. Four kinds of spectra (absorption, emission, emission polarization, and excitation polarization) measured on C-phycocyanin isolated from the cyanobacterium Mastigocladus laminosus were used as the experimental data set. Without any assumption of spectral shape, the absorption and fluorescence spectra of both chromophores were unambiguously resolved and their fluorescence quantum yields were evaluated. By combining the spectra of the alpha-subunit, independently measured, with the resolved spectra of the beta-subunit, the fluorescence and fluorescence polarization spectra and the fluorescence quantum yield of the monomer were estimated; they agree with experimental values to within an acceptable error. Further, the matrix of energy transfer rates in the monomer was estimated; it gave a significantly different result (by up to 40%) from previously estimated ones.     

Biliproteins and phycobilisomes from cyanobacteria and red algae at the extremes of habitat

This review considers the properties of biliproteins from cyanobacteria and red algae that grow in extreme habitats. Three situations are presented: cyanobacteria that grow at high temperatures; a red alga that grows in acidic conditions at high temperature; and an Antarctic red alga that grows in the cold in dim light conditions. In particular, the properties of their biliproteins are compared to those from organisms from more usual environments. C-phycocyanins from two cyanobacteria able to grow at high temperatures are found to differ in their stabilities when compared to C-phycocyanin from mesophilic algae. They differ in opposite ways, however. One is more stable to dissociation than the mesophilic protein, and the other is more easily dissociated at low temperatures. The thermophilic proteins resist thermal denaturation much better than the mesophilic proteins. The most thermophilic cyanobacterium has a C-phycocyanin with a unique blue-shifted absorption maximum which does not appear to be part of the adaptation of the cyanobacterium to high temperature. The C-phycocyanin from the high-temperature red alga is able to resist dissociation better than mesophilic C-phycocyanins. Electron micrographs show the phycobilisomes of these algae. The Antarctic alga grows under ice at some distance down the water column. Its R-phycoerythrin has a novel absorption spectrum that gives the alga an improved ability to harvest blue light. This may enhance its survival in its light-deprived habitat.     

Long-lived tryptophan fluorescence in phosphoglycerate mutase

Phosphoglycerate mutase (PGM; EC 2.7.5.3) isolated from rat and rabbit muscle has been shown to possess an unusually long-lived fluorescence component when excited by ultraviolet light below 310 nm. On the basis of spectral and physical measurements, this 16.4 (+/- 0.2) ns fluorescence lifetime at room temperature is assigned to a tryptophan residue in an unusual environment. The emission profile of this long-lived tryptophan is red shifted from the other tryptophans of PGM by approximately 25 nm. PGM has been crystallized and sequenced from yeast where it has been shown to be a tetramer with 29K subunits. However, we have not been able to detect the existence of an unusually long-lived fluorescence component in the yeast isomer. The long fluorescence lifetime is lost upon denaturation of rabbit PGM and is partially restored upon introduction of the protein to a nondenaturing environment, suggesting the long lifetime is not the result of a covalent modification. The PGM molecule was studied by a number of techniques including time-resolved tryptophan fluorescence, quenching studies of tryptophan fluorescence, and enzyme activity studies. The long-lived fluorescence has been shown to be statistically quenched by Br-, I-, and Cu2+ in the submillimolar region while the acrylamide quenching shows the tryptophan is marginally accessible to solvent. Characterization of the long-lived fluorescence and its possible sources are discussed.     

Increased aggregation of C-phycocyanin produced by phenol and benzene

The effect of selected aromatic compounds on aggregates of C-phycocyanin was examined by analytic ultracentrifugation. Benzene and phenol caused an increase in aggregation. The principal change, a conversion of 6S to an 11S species, is postulated to result from the interaction of benzene and phenol with hydrophobic areas involved in protein-protein contact.The C-phycocyanin used in these studies included that extracted from Spirulina platensis, a Cyanophyta that grows in a highly alkaline environment. The pH dependence of the aggregation properties of this protein, however, was similar to that of C-phycocyanins from algae grown in normal environments. This suggests that the internal biopolymers of organisms which naturally exist at extremes of pH are somehow protected from exposure to these extremes.     

The characterization of C-phycocyanin from an extremely halo-tolerant blue–green alga, Coccochloris elabens

C-Phycocyanin was isolated and purified from a uni-algal culture of an extremely halo-tolerant blue-green alga, Coccochloris elabens. This alga can be grown under laboratory conditions in 25% (w/v) NaCl. Purified halophile phycocyanin was characterized by amino acid analysis and the measurement of sedimentation velocity, fluorescence polarization and immunodiffusion as a function of protein concentration, pH and ionic strength. The results were compared with those of studies of phycocyanin isolated from Plectonema calothricoides and from several other sources. The states of aggregation previously characterized as being present in other C-phycocyanins, monomer, trimer and hexamer, were present in halophile phycocyanin and were characterized as antigenically related to all C-phycocyanins tested. The equilibrium between 3S monomer and 11S hexamer at low concentrations in halophile phycocyanin was quantitatively similar to that for other phycocyanins. The effect of pH and ionic strength on the 6S (trimer) and 11S (hexamer) aggregation of halophile phycocyanin was markedly salt-dependent and the relative amount of each aggregate in the presence of 2m-NaCl was like that of C-phycocyanin from mesophiles, in the absence of additional salt. In antigenic relationship and aggregation properties, the phycocyanin from C. elabens appeared to be most closely related to that isolated from the thermophilic blue-green alga, Synechococcus lividus. Amino acid content of the halophile phycocyanin indicated the presence of a significantly larger number of acidic residues than that found in mesophiles. Explanations of the properties of the halophile protein require consideration of a strong contribution of hydrophobic forces and utilize both charge-shielding and salting-out effects.     

Studies on C-phycocyanin from Cyanidium caldarium, a eukaryote at the extremes of habitat

C-Phycocyanin, a biliprotein, was purified from the red alga, Cyanidium caldarium. This alga grows at temperatures up to 57 degrees C, a very high temperature for a eukaryote, and at pH values down to 0.05. Using the chromophores on C-phycocyanin as naturally occurring reporter groups, the effects of temperature on the stability of the protein were studied by circular dichroism and absorption spectroscopy. The protein was unchanged from 10 to 50 degrees C, which indicates that higher temperatures are not required to cause the protein to be photosynthetically active. At 60 and 65 degrees C, which are above the temperatures at which the alga can survive, the protein undergoes irreversible denaturation. Gel-filtration column chromatography demonstrated that the irreversibility is caused by the dissociation of the trimeric protein to its constitutive polypeptides. Upon cooling, the alpha and beta polypeptides did not reassemble to the trimer. Unlike phycocyanins 645 and 612, the C-phycocyanin does not show a reversible conformational change at moderately high temperatures. At constant temperature, the C-phycocyanin was more stable than a mesophilic counterpart. It is designated a temperature-resistant protein.     

Effect of Cu2+ on PSⅡ Activity and Energy Transfer of Phycobillisome in Intact Cells of Spirulina platensis

Addition of Cu2+ at low concentrations, to intact cells of the cyanobacterium, Spirulina platensis, at room temperature, caused an enhancement in intensity of fluorescence emitted by phycocyanin and induced a blue shift at the emission peak, both of which indicated changes in energy transfer within the phycobillisomes. Cu2+ also suppressed the whole-chain electron transport activity (H2O→MV) and water-splitting activity of the photosystem Ⅰ. When isolated phycocyanin and allophycocyanin were exposed to very low concentrations of Cu2+ ions, C-phycocyanin but not allophycocyanin, exhibited decrease not only in the absorbance in the longer wavelength (616--620 nm) region, but also in the fluorescence emission intensity at 647 nm accompanied by a blue shift to 643 nm. These results suggested that Cu2+ selectively bleach C-phycocyanin.     

The chromophore of phytochrome

The chromophore of phytochrome, the plant photomorphogenic pigment, was cleaved from the associated protein. Chromatographic and spectral properties indicated that it was a bilitriene closely similar to but distinct from the chromophore of C-phycocyanin and allophycocyanin.     

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.     

Isolation and characterization of disc-shaped phycobilisomes from the red alga rhodella violacea

Disc-shaped phycobilisomes were purified from Triton X100 treated cell homogenates of the unicellular marine red alga, Rhodella violacea. Their absorption spectrum had principal maxima at 544 and 568 nm (B-phycoerythrin), 624 nm (C-phycocyanin) and a distinct shoulder at 652 nm (allophycocyanin). Intermolecular energy transfer within the phycobilisomes was clearly demonstrated by fluorescence data. Excited at 546 nm intact phycobilisomes showed a main fluorescence emission maximum at 665 nm, a minor one at 577 nm and a shoulder at 730 nm.Dissociated phycobilisomes revealed a composition of 58% B-phycoerythrin, 25% C-phycocyanin and 17% allophycocyanin under the cultural conditions used. Analytical methods resolved no other components than phycobiliproteins. In addition to the defined C-phycocyanin and two isoproteins of B-phycoerythrin a stable heterogeneous aggregate of B-phycoerythrin/C-phycocyanin was separated in considerable amounts.In the electron microscope negatively stained phycobilisomes appeared as elliptical aggregates having dimensions slightly above the values found in ultrathin sections and a detailed subunit structure. All observations and data suggest a new rhodophytan phycobilisome type in Rhodella violacea.Abbreviations PBS phycobilisome(s) - PE B-phycoerythrin - PC C-phycocyanin - APC allophycocyanin - C concentration (mg/ml) - E extinction     

Interaction of Pb(II) ions with C-phycocyanin from Spirulina platensis: effect of ionic strength

The effect of Pb(II) ions on C-phycocyanin from Spirulina platensis was studied by the method of fluorescence spectroscopy. The efficiency of quenching was analyzed using the Stern-Volmer relation. Isotherms of adsorption were constructed, and the constants of binding of Pb(II) ions to C-phycocyanin in solutions of varying ionic strength were determined by equilibrium dialysis and atomic absorption spectroscopy. The constants of binding of Pb(II) ions to C-phycocyanin in 2, 20, and 50 mM NaN03 solutions were estimated to be 4.79 x 10(5), 3.63 x 10(5) and 2.82 x 10(5), respectively. It was found that the interaction of Pb(II) ions with C-phycocyanin has a cooperative character at all values of ionic strengths studied.     

Picosecond fluorescence of cryptomonad biliproteins. Effects of excitation intensity and the fluorescence decay times of phycocyanin 612, phycocyanin 645, and phycoerythrin 545.

The fluorescence of purified biliproteins (phycocyanin 645, phycocyanin 612, and phycoerythrin 545) from three cryptomonads, Chroomonas species, Hemiselmis virescens, and Rhodomonas lens, and C-phycocyanin from Anacystis nidulans has been time resolved in the picosecond region with a streak camera system having less than or equal to 2-ps jitter. The fluorescence lifetimes of phycocyanins from Chroomonas species and Hemiselmis virescens are 1.5 +/- 0.2 ns and 2.3 +/- 0.2 ns, respectively, regardless of the fluence of the 30 ps, 532-nm excitation pulse. (Fluence [or photons/cm2] = f intensity [photons/cm2s]dt.). In contrast, that of C-phycocyanin is 2.3 +/- 0.2 ns when the excitation fluence is 8.2 X 10(11) photons/cm2 and decreases to a decay approximated by an exponential decay time of 0.65 +/- 0.1 ns at 7.2 X 10(16) photons/cm2. The cryptomonad phycoerythrin fluorescence decay lifetime is also dependent on intensity, having a decay time of 1.5 +/- 0.1 ns at low fluences and becoming clearly biphasic at higher fluences (greater than 10(15) photons/cm2). We interpret the shortening of decay times for C-phycocyanin and phycoerythrin 545 in terms of exciton annihilation, and have discussed the applicability of exciton annihilation theories to the high fluence effects.     

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.     

The observation of structural transitions of a single protein molecule

Coherent neutron scattering measurements of an amorphous, in vivo deuterated C-phycocyanin are compared with a calculation of the individual protein molecule's coherent static structure factor. Both show the significant features associated with known structure factors of several amorphous materials, most notably, an unusually sharp first diffraction peak occurring near 1.4 A(-1). We show that in the protein, such a peak results from the product of a form factor associated with correlations of atoms within individual amino acids and a structural term expressing inter-amino-acid correlations. The measurement, interpreted through behavior of the first diffraction peak, indicates that inter-amino-acid correlations - a measure of the protein's medium-range structure - undergo transitions which are primarily related to hydration rather than to temperature.     

Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bed adsorption chromatography

C-phycocyanin was purified on a large scale by a combination of expanded bed adsorption, anion-exchange chromatography and hydroxyapatite chromatography from inferior Spirulina platensis that cannot be used for human consumption. First, phycobiliproteins were extracted by a simple, scaleable method and then were recovered by Phenyl-Sepharose chromatography in an expanded bed column. The purity (the A(620)/A(280) ratio) of C-phycocyanin isolated with STREAMLINE column was up to 2.87, and the yield was as high as 31 mg/g of dried S. platensis. After the first step, we used conventional anion-exchange chromatography for the purification steps, with a yield of 7.7 mg/g of dried S. platensis at a purity greater than 3.2 and with an A(620)/A(650) index higher than 5.0. The fractions from anion-exchange chromatography with a level of purity that did not conform to the above standard were subjected to hydroxyapatite chromatography, with a C-PC yield of 4.45 mg/g of dried S. platensis with a purity greater than 3.2. The protein from both purification methods showed one absolute absorption peak at 620 nm and a fluorescence maximum at 650 nm, which is consistent with the typical spectrum of C-phycocyanin. SDS-PAGE gave two bands corresponding to 21 and 18 kDa. In-gel digestion and LC-ESI-MS showed that the protein is C-phycocyanin.     

Picosecond study of energy-transfer kinetics in phycobilisomes of Synechococcus 6301 and the mutant AN 112

Excitation-energy-transfer kinetics in isolated phycobilisomes from the cyanobacterium Synechococcus 6301 (Anacystis nidulans) and the mutant AN 112 (rods containing one hexameric C-phycocyanin unit only) was investigated by picosecond absorption and fluorescence techniques. The different chromophores in the phycobilisomes were selectively excited. A lifetime component of about 10 ps was found for both C-phycocyanin and allophycocyanin in both types of phycobilisomes. We assign these signals to a transfer of excitation energy from sensitizing (‘s’) to fluorescing (‘f’) chromophores within C-phycocyanin and allophycocyanin units. A 10 ps component was also observed in the anisotropy relaxation measurements. The anisotropy decay is attributed mainly to differently oriented transition dipole moments of ‘s’- and ‘f’-chromophores and partially to ‘f’ → ‘f’ transfer. An absorption recovery signal of τ ≈ 90 ps at λ ≤ 630 nm in phycobilisomes of Synechococcus 6301 is reduced to 40–50 ps in AN 112 phycobilisomes. This is rationalized in terms of a decreased rod → core transfer time in the shorter rods of AN 112. The 40–50 ps lifetime of fluorescence and absorption recovery in AN 112 phycobilisomes is assigned mainly to a rate-limiting transfer step between C-phycocyanin and the allophycocyanin core. A decay component of allophycocyanin τ ≈ 50 ps was observed both in absorption recovery measurements and in fluorescence decay. It is assigned to energy transfer to the terminal chromophores. The final emitter(s) of the phycobilisomes from AN 112 have fluorescence lifetimes of 1.9 and 1.3 ns. We find a good correlation in the fluorescence kinetics between the decay times of phycocyanin and allophycocyanin and the fluorescence risetimes of the terminal emitters.     

Comparative study of recombinant rat nucleoside diphosphate kinases alpha and beta by intrinsic protein fluorescence

Nucleoside diphosphate (NDP) kinases of mammals are hexamers of two sorts of randomly associated highly homologous subunits of 152 residues each and, therefore exist in cell as NDP kinase isoforms. The catalytic properties and three-dimensional structures of the isoforms are very similar. The physiological meaning of the existence of the isoforms in cells remained unclear, but studying recombinant rat NDP kinases alpha and beta, each containing only one sort of subunits, we discovered that, in contrast to the isoenzyme beta, NDP kinase alpha is able to interact with the complex between bleached rhodopsin and G-protein transducin in retinal rod membranes at lowered pH values (Orlov et al. FEBS Lett. 389, 186-190, 1996). In order to search for possible molecular basis of such differences between these isoenzymes, a detailed comparative study of their intrinsic fluorescence properties in a large range of solvent conditions was performed in this work. The isoenzymes alpha and beta both contain the same three tryptophan (Trp78, 133, Ind 149) and four tyrosine (Tyr 52, 67, 147, and 151) residues per subunit, but exhibit pronounced differences in their fluorescence properties (both in spectral positions and shape and quantum yield values) and behave differently under pH titration. Whereas NDP kinase alpha undergoes spectral changes in the pH range 5-7 with the mid-point at 6.2, no unequivocal indication of a structural change of NDP kinase beta under pH titration from 9 to 5 was obtained. Since the pH dependencies obtained for fluorescence of isoenzyme alpha resembles the dependence of its binding to the rhodopsin-transducin complex it was suggested that the differences between the NDP kinase isoenzymes alpha and beta in the pH-induced behavior, revealed by the fluorescence spectroscopy, and the differences in their ability to interact with rhodopsin-transducin complex may have the same physical nature, that would be a physico-chemical reason of possible functional dissimilarity of NDP kinase isoforms in cell. An additional analysis of three-dimensional structure of homologous NDP kinases revealed that the source of the differences in fluorescence properties and pH-titration behavior between the isoenzymes alpha and beta may be due to the difference in their global electrostatic charges, rather than to any structural differences between them at neutral pH. The unusually high positive electrostatic potential at he deeply buried active site Tyr52 makes possible that it exists in deprotonated tyrosinate form at neutral and moderately acidic solution. Such a possibility may account for rather unusual fluorescence properties of NDP kinase alpha: (i) rather long-wavelength emission of NDP kinase alpha at ca. 340 nm at pH ca. 8 at extremely low accessibility to external quenchers and, possibly, (ii) an unusually high quantum yield value (ca. 0.42).     

Interaction of C-phycocyanin with lipid monolayers under nitrogen and in the presence of air

The surface interaction of C-phycocyanin with lipids was studied using the monolayer technique. The surface activity of the protein was found to be higher at the lipid-water interface than at the nitrogen-water interface, particularly at high surface pressures of the lipid monolayer. The maximum initial surface pressures beyond which phycocyanin could not penetrate the dipalmitoylphosphatidylcholine and monogalactosyldiglycerol monolayers were 27 and 30 mN m-1, respectively. Below these values the protein demonstrated preferential interaction with the monogalactosyldiglycerol monolayer. The surface properties of the unfolded protein at pH 2.5 at the lipid-water interface were compared with those of the protein at pH 7.0. Higher affinity of the three-dimensional structure of the protein to lipid monolayers was observed, in particular by high subphase protein concentration. When the lipid films were subjected to oxidation stress by exposure to air, the surface properties of C-phycocyanin and dipalmitoylphosphatidylcholine were not greatly affected but the surface activity of monogalactosyldiacylglycerol was reduced dramatically by autoxidation. The oxidation of monogalactosyldiacylglycerol could not be prevented by the introduction of C-phycocyanin molecules at the lipid-water interface.