Phycobiliproteins Phycoerythrin

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 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. 

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). 

Despite these problems, flow cytometry has had some noted success in aquatic research, particularly in relation to studies on the phytoplankton. Because all phytoplankton possess chlorophyll, but only the cyanobacteria possess the phycobiliproteins, autofluorescence signatures from water samples, based on the chlorophyll (fluorescence >630 nm), phycoerythrin (fluorescence <590 nm), and forward scatter of particles, have been used to characterize the changes that occur in plankton at different depths or at different locations (Figs. 11.5 and 11.6). 

In the PBS rods the phycobiliprotein hexamers can be identified by high-resolution electron microscopy as discs, subdivided into two halfs (a/3-trimers) of 30 A thickness [80,143]. Deeper insight into the molecular structure of the trimers and hexamers was achieved by X-ray crystallographic analyses of biliproteins. In the last century, strikingly coloured phycocyanin and phycoerythrin crystals had already been observed by Molish [144]. Recently, several C-phycocyanins [145-147], B-phycoerythrin [147,148] and phycoerythrocyanin [149] have been crystallized... 

Phycobiliproteins, such as phycocyanin and phycoerythrin, are members of a family of fluorescent accessory, nonchlorophyll-based pigments found in cyanobacteria and eukaryotic algae. The phycobiliproteins have characteristic broad absorption profiles spanning 450-600 nm, emissions ranging 570-660 nm, and small stokes shifts see Fig. 3. The major structural subunits, phycoerythrobilin (PEB) or phycocyanobilin... 

Figure 3 Absorption and emission profile of the phycobiliprotein B-phycoerythrin (B-PE), which is a multi-subunit multi-chromophore fluorescent protein with exceptional absorption and emission properties.

The two major polypeptides making up each phycobiliprotein are the so-called a- and p-subunits, with molecular weights of 17 and 18 kDa, respectively, and each consisting of 160-180 amino acids. Some phycobiliproteins, the phycoerythrins, contain an additional, 30-kDa y-subunit. 

Table 1. Examples drawn from four classes of phycobiliproteins, allophycocyanin (APC), phycocyanin (PC), phycoerythrocyanin (PEC), and phycoerythrin (PE), arranged in the order of decreasing wavelengths of their major absorption bands. and x " are the peak wavelengths of the principal (and minor) absorption and fluorescence bands, respectively. See List of Abbreviations for the full names of the different phycobiliproteins. Table adapted from Glazer (1982) Phycobilisomes Structure and dynamics. Annu Rev Microbiology 36 178 and Glazer (1989) Light guides. Directionai energy transfer in a photosynthetic antenna. J Biol Chem. 264 2.

The absorption and fluorescence spectra ofthe four representative phycobiliproteins are shown in Fig. 5 allophycocyanin from the filamentous cyanobacterium Ana aena variabilis, R-pbycocyanin and B-phycoerythrin from tbe unicellular red alga Porphyridium cruentum, and R-phycoerytbrin from the red alga Gastroclonium coulteri. 

Other dyes such as dansyl and AEDANS are popular donors in protein studies, in part because of the relatively long lifetimes (13-20 nsec), large (150 nm) Stokes shifts, and reasonable quantum yields (0.1-0.5). If steric hindrance is not a problem and a large-sized donor or acceptor can be used, the multichromophoric phycobiliproteins (molecular weight of 104,000 for B- or R-phycoerythrin 240,000 for allophycocyanin) make excellent donors or acceptors, having extinction coefficients which can exceed... 

The phycobiliproteins are hard to beat as extremely bright antibody labels for fluorescence analysis of cell surface antigens by flow cytome-try 15,16 These bacterial photosynthetic macromolecules each have up to 34 individual bilin fluorophores wrapped within the polypeptide structure. An example is R-phycoerythrin (R-PE), which has an extinction coefficient of 2 X 10 L/mol cm and a quantum yield of 0.68. Usually there is room for only one PE molecule per antibody, because the size of an R-PE label is 1.5 times that of an IgG antibody. The large size of the complex reduces the kinetics of binding to cell surface antigens, and some intracellular markers are inaccessible to the R-PE-labeled antibody. For intracellular measurements, lower molecular mass fluorophores (<1 kDa) are usually preferred. 

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