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.     

Molecular weight and shape of the phycocyanin hexamer

The hexamer of phycocyanin from Phormidium luridum has been isolated and purified by ammonium sulfate fractionation and gel chromatography. The protein is characterized by the sedimentation constant S°_{20, w} = 10.2S, the diffusion coefficient D_{20, w} = 4.73 × 10^?7 cm²/sec, and intrinsic viscosity [η] = 3.89 ml/g. The molecular weight of the aggregate is 209,000. The shape and dimensions of the hexamer are discussed in terms of a model consisting of subunits arranged with C₆ symmetry. The monomers, assumed to be spherical, are found to have a radius of 22 Å, and the diameter across the hexamer is 132 Å. The latter figure agrees closely with dimensions observed in electron micrographs.     

Association and dissociation of phycocyanin by small molecules

Sedimentation velocity experiments showed that tetraalkylammonium salts, with alkyl chain lengths ranging from methyl to pentyl and in the concentration range from 0.02 to 0.16 m, decrease the aggregation of C-phycocyanin in sodium phosphate buffers, pH 6.0 and 7.0, I 0.1, at 23 °C.Tetrabutylammonium and tetrapentylammonium bromides and chlorides disaggregate 11S and 17S C-phycocyanin aggregates to produce a 6S subunit. The larger alkyl group produces the greater effect. An explanation for this effect would be that hydrophobic and possibly ionic interactions between the protein and the tetraalkylammonium cation effectively block assembly of the 6S to 11S aggregate. These salts may provide a novel means for the control of protein assembly.Recent studies on the effect of neutral aromatic compounds on C-phycocyanin aggregation were extended to saturated solutions of α- and β-naphthol, with concentrations of <0.003 and <0.006 m, respectively. Sedimentation velocity experiments and absorption spectroscopy suggest that the aggregation of C-phycocyanin is increased by naphthol binding to the protein.     

High-resolution crystal structures of trimeric and rod phycocyanin

The phycobilisome light-harvesting antenna in cyanobacteria and red algae is assembled from two substructures: a central core composed of allophycocyanin surrounded by rods that always contain phycocyanin (PC). Unpigmented proteins called linkers are also found within the rods and core. We present here two new structures of PC from the thermophilic cyanobacterium Thermosynechococcus vulcanus. We have determined the structure of trimeric PC to 1.35 Å, the highest resolution reported to date for this protein. We also present a structure of PC isolated in its intact and functional rod form at 1.5 Å. Analysis of rod crystals showed that in addition to the α and β PC subunit, there were three linker proteins: the capping rod linker (L_R^8.7), the rod linker (L_R), and only one of three rod-core linkers (L_RC, CpcG4) with a stoichiometry of 12:12:1:1:1. This ratio indicates that the crystals contained rods composed of two hexamers. The crystallographic parameters of the rod crystals are nearly identical with that of the trimeric form, indicating that the linkers do not affect crystal packing and are completely embedded within the rod cavities. Absorption and fluorescence emission spectra were red-shifted, as expected for assembled rods, and this could be shown for the rod in solution as well as in crystal using confocal fluorescence microscopy. The crystal packing imparts superimposition of the three rod linkers, canceling out their electron density. However, analysis of B-factors and the conformations of residues facing the rod channel indicate the presence of linkers. Based on the experimental evidence presented here and a homology-based model of the L_R protein, we suggest that the linkers do not in fact link between rod hexamers but stabilize the hexameric assembly and modify rod energy absorption and transfer capabilities.     

The photoregulated expression of multiple phycocyanin species. A general mechanism for the control of phycocyanin synthesis in chromatically adapting cyanobacteria

The regulation of phycocyanin synthesis in response to growth in chromatic illumination was studied in 69 strains of cyanobacteria. Cyanobacteria (24 of 31 strains examined), which chromatically adapt by modulating the synthesis of both phycocyanin and phycoerythrin, controlled phycocyanin synthesis through the differential, photoregulated expression of two phycocyanin species (two alpha-type and two beta-type subunits). For these strains the expression of one pair of phycocyanin subunits was constitutive (i.e. irrespective of the light wavelength in which the cells were grown); the expression of the second pair of phycocyanin subunits occurred specifically during growth in red light. Two facultatively heterotrophic cyanobacteria, Calothrix strains 7101 and 7601, synthesized both the constitutive and the inducible pairs of phycocyanin subunits when grown heterotrophically in the dark after transfer from either red or green light. No evidence for the existence of multiple and/or photoregulated phycocyanin species was found for cyanobacteria (25 strains) incapable of chromatic adaptation, nor for cyanobacteria (13 strains) which chromatically adapt by modulating the synthesis of phycoerythrin alone.     

Method for efficient extraction of phycocyanin from Spirulina

Summary Of several different methods tried for the extraction of phycocyanin (PC) from the cyanobacterium Spirulina, treatment with 1800 Units / ml lysozyme and 100 ppm linear alkyl benzene sulfonate afforded good PC yield with high specific content, resulting in 66 and 98% recovery of PC, respectively.     

Amoebic grazing of freshwater Synechococcus strains rich in phycocyanin

Fifteen strains of naked amoebae were presented with 19 strains of Synechococcus on an agar surface. After 14 days of incubation, each of the 285 combinations yielded one of three responses. 42.1% of combinations showed clearing (digestion) of the Synechococcus (C), 56.5% of combinations showed no clearing of the Synechococcus (N) while 1.4% of combinations showed partial clearing of the Synechococcus (P). In general, the Synechococcus strains showed variability in their susceptibility to digestion by the amoebae and the amoebae showed variability in their ability to digest the Synechococcus strains. There was no evidence for amoebae actively selecting profitable prey and equivalent-sized Synechococcus strains were ingested at the same rate, irrespective of their fate. There was some evidence of 'size-selective' grazing in that amoebae ingested the smaller Synechococcus strains at higher rates than the larger strains. However, there was no correlation between prey size and their ultimate fate. These data suggest that amoebae are not selective with regard to the ingestion of synechococci, but that 'selection' occurs at the digestion stage, i.e. whether the synechococci are digested or not.     

An estimation of the dimensions of the phycocyanin molecule

Knowledge of the total particle volume and the specific volumes of the constituent polar and nonpolar amino acid residues of a globular protein may be used in suitable instances to estimate the size and shape, of the macromolecule. Use has been made of the data, which is available in the literature for phycocyanin from Plectonema calothricoidcs, to predict possible models for the monomer and hexamer forms of this protein. The monomer is well represented by a prolate ellipsoid with semiaxes of 45 and 17Å and an approximate molecular weight of (46000). The hexamer is envisaged as the aggregate of six such monomers, in juxtaposition, with their major axes parallel so as to form a closed ring structure of about 268000 molecular weight. These proposed models are consistent with the previously published electron micrographs and hydrodynamic properties of this protein.     

Chemical stabilization of the phycocyanin from cyanobacterium Spirulina platensis

Phycocyanin (PC) prepared from a cyanobacterium Spirulina platensis by the DEAE-DE52 cellulose column chromatography that was developed by gradient elution of 50-250 mM phosphate buffer (pH 7.0) was stabilized by its subunits cross-linked covalently with formaldehyde. The single blue band that the chemically stabilized PC showed in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the stabilized PC still maintained its trimeric aggregate form even after its incubation at 60 degrees C for 3h and at 100 degrees C for 10 min in the denatured buffer containing 5% (w/v) SDS. Moreover, the stabilized PC exhibited similar spectroscopic properties of absorption and fluorescence to those of the native PC, and showed adequate energy coupling with R-phycoerythrin (R-PE) after it was conjugated with R-PE via glutaraldehyde.     

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.