Phycoerythrins

Algae are classified according to their colors (1) Chlorophyceae (green), (2) Rhodophyceae (red), (3) Cyanophyceae (bine-green), and (4) Pheophyceae (brown). The major pigments inclnde chlorophylls a, b, and c, P-carotene, phycocyanin, xanthophylls, and phycoerythrin. All these pigments have great potential for applications in foods, pharmacenticals, and cosmetics. 

The phycobiliproteins are accessory photosynthetic pigments aggregated in cells as phycobilisomes that are attached to the thylakoid membrane of the chloroplast. The red phycobiliproteins (phycoerythrin) and the blue phycobiliprotein (phycocy-anin) are soluble in water and can serve as natural colorants in foods, cosmetics, and pharmaceuticals. Chemically, the phycobiliproteins are built from chro-mophores — bilins — that are open-chain tetrapyrroles covalently linked via thio-ether bonds to an apoprotein. ... 

Porphyridium species are the sources of fluorescent pink color. The main Porphyridium phycobiliproteins are B-phycoerythrin and b-phycoerythrin. Maximum absorbance of a 1% solution of B-phycoerythrin in a 1-cm cuvette is at 545 inn, and the fluorescence emission peak is at 575 inn molecular weight is 240 kda. Batch culture of Porphyridium species outdoors yields approximately 2(X) mg of colorant per liter of culture after 3 days the phycoerythrin level in the colorant is about 15%. A higher concentration of phycoerythrin, up to 30%, can be achieved under optimal algal culture conditions. 

The red microalga Porphyridium aerugineum is a source of blue color. This species is different from other red microalgae in that it lacks red phycoerythrin and its phycocyanin is C-phycocyanin rather than the R-phycocyanin that accompanies phycoerythrin found in many red algae and in other Porphyridium species. However, the biochemicals produced by P. aerugineum are similar to those of other red microalgae, e.g., sulfated polysaccharides, carotenoids, and lipids. An alternative source of C-phycocyanin is Spirulina platensis. ... 

Roman, R.B. et al.. Recovery of pure B-phycoerythrin from the microalga Porphy-ridium cruentum, J. BiotechnoL, 93, 73, 2002. 

Bermejo, R. et al.. Preparative purification of B-phycoerythrin from the microalga Porphyridium cruentum by expanded-bed adsorption chromatography, J. Chromatogr. B, 790, 317, 2003. 

Glazer, A.N. and Hixson, C.S., Subunit structure and chromophore composition of rhodophytan phycoerythrins Porphyridium cruentum B-phycoerythrin and b-phyco-erythrin, J. Biol. Chem., 252, 32, 1977. 

Mooradian (1993) has studied the antioxidant properties of 14 steroids in a non-membranous system in which the fluorescence of the protein phycoerythrin was measured in the presence of a lipid peroxyl radical generator (ABAP). Oxidation of the protein produces a fluorescent species. Quenching of fluorescence by a test compound indicates antioxidant activity. Oestrone, testosterone, progesterone, androstenedione, dehydroepian-drosterone, cortisol, tetrahydrocortisone, deoxycorti-... 

The participation of the phycobiliproteins in the absorption ofphotokinetically active light has been demonstrated above. Peaks of around 565 and 615 nm in the action spectra indicate the involvement of C-phycoerythrin andC-phycocanin. These pigments transfer energy to the reaction center of PS II and suggest the participation of the non-cyclic electron transport and coupled phosphorylation. 

Phototactic action spectra of Phormidium autumnale and Phormidium uncinatum, measured by Nultsch86>89), show prominent maxima in the absorption range of C-phycoerythrin and smaller, but distinct, peaks in the absorption range of C-phyco-cyanin. Red light absorbed by chlorophyll a is not active, while in the blue range absorbedby the Soret band, the action spectrum shows aminimum(Fig. 6). Nultsch87) concluded that biliproteins are photoreceptors of phototaxis, but independently of the photosynthetic electron transport and phosphorylation. 

Fig. 6. Phototactic action spectrum (circles and heavy solid line) of Phormidium uncinatum. For comparison the absorption spectra of C-phycoerythrin (fine solid line) and myxoxanthophyll (dashed-dotted line) are drawn. Abscissa wavelength in nm Ordinates phototactic reaction value Rt in relative units and absorbance respectively (modified after Nultsch89))...

The action spectmm of positive and negative phototaxis of Anabaena variabilis was measured recently106). This species contains no C-phycoerythrin. Accordingly, maximum activity is found at around 615 nm (Fig. 7). In addition, in this form a second maximum occurs at around 675 nm, and a third small, but distinct, one at 440 nm, both indicating that chlorophyll a is also involved in the active light absorption (see above). The utilization via photosynthesis, however, could be excluded in this case, since the trichomes oriented themselves perfectly well to the light direction in the presence of photosynthetic inhibitors, such as DCMU and DBMIB, at concentrations in which the photosynthetic oxygen evolution was almost completely inhibited. 

Fig. 8. Difference spectrum (green irradiated minus red irradiated) of phycochrome c (solid line), action spectrum for induction of filamentous growth of Nostoc muscorum (dotted line), and action spectrum for inhibition of C-phycoerythrin synthesis in Tolypothrix tenuis (dashed-dotted line). Abscissa wavelength in nm Ordinates relative absorbance change und relative action (after Bjorn and Bjorn 5))...

When the trap light is utilized predominantly by photosystem II (e.g., by irradiation of 563 nm, which is absorbed by the main accessory pigment C-phycoerythrin),... 

There are three main classes of phycobiliproteins, differing in their protein structure, bilin content, and fluorescent properties. These are phycoerythrin, phycocyanin, and allo-phycocyanin (APC). There are two main forms of phycoerythrin proteins commonly in use B-phycoerythrin isolated from Porphyridium cruentum and R-phycoerythrin from Gastroclonium coulteri. There also are three main forms of pigments found in these proteins phycoerythrobilin, phycourobilin, and phycocya no bilin (Glazer, 1985). The relative content of these pigments in the phycobiliproteins determines their spectral properties. All of them,... 

The spectral properties of four major phycobiliproteins used as fluorescent labels can be found in Tables 9.1 and 9.2. The bilin content of these proteins ranges from a low of four prosthetic groups in C-phycocyanin to the 34 groups of B- and R-phycoerythrin. Phycoerythrin derivatives, therefore, can be used to create the most intensely fluorescent probes possible using these proteins. The fluorescent yield of the most luminescent phycobiliprotein molecule is equivalent to about 30 fluoresceins or 100 rhodamine molecules. Streptavidin-phycoerythrin conjugates, for example, have been used to detect as little as 100 biotinylated antibodies bound to receptor proteins per cell (Zola et al., 1990). 

In addition to the wide range of commercial probes, many other fluorescent molecules have been synthesized and described in the literature. Only a handful, however, are generally used to label antibody molecules. Perhaps the most common fluorescent tags with application to immunoglobulin assays are reflected in the main derivatives produced by the prominent antibody manufacturing companies. These include derivatives of cyanine dyes, fluorescein, rhod-amine, Texas red, aminomethylcoumarin (AMCA), and phycoerythrin. Figure 20.16 shows the reaction of fluorescein isothiocyanate (FITC), one of the most common fluorescent probes, with an antibody molecule. 

---