Phycocyanin, fluorescence

Seppala, J., Yldstalo, R, Kaitala, S., Hallfors, S., Raateoja, M., Maunula, P, 2007. Ship-of-opportunity based phycocyanin fluorescence monitoring of the filamentous cyanobacteria bloom dynamics in the Baltic Sea. Estuarine Coastal and Shelf Science, 73, 489-500. 

The algal extract of P. aerugineum is blue, with maximum absorbance at a wavelength of 620 nm and a red fluorescence with maximum emission at 642 nm. The main phycobiliprotein, C-phycocyanin, is the same type of phycocyanin found in most Cyanobacteria. The chromophores are composed of phycocyanobilins, conjugated to an apoprotein via thioether bonds. 

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). 

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 1. Fluorescence spectral signatures for different algal color groups (E = excitation F = fluorescence). Key to schemes 1, diatoms and dinofla-gellates la green algae 2, phycoerythrin of cyanobacteria 3, phycocyanin. (Reproduced with permission from Ref. 5. Copyright 1981 Burgess Publishing Company.)...

Figure 3. Absorption and fluorescence spectral signature of cyanobacteria Synechococcus sp. (Bigelow Laboratory, Clone L1601). Note the fluorescence emission of phycocyanin at longer wavelengths.

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.

Fig. 5. Absorption and fluorescence emission spectra of isolated allophycocyanin, R-phycocyanin, B-phycoerythrin and R-phycoeryth-rin. Figure source Glazer and Stryer (1984) Phycofluorprobes. Trends in Biochem Sci 9 2.

Fremy E (1860) Recherches sur la matiere colorante verte des feuilles. Compt Rend 50 405-412 French CS and Young VMK (1952) The fluorescence spectra of red algae and the transfer of energy from phycoerythrin to phycocyanin and Chi. J Gen Physiol 35 873-890 Gaffron H and Wohl K (1936) Zur Theorie der Assimilation. 

Figure B2.1.7 Transient hole-burned spectra obtained at room temperature with a tetrapyrrole-containing light-harvesting protein subunit, the a subunit of C-phycocyanin. Top fluorescence and absorption spectra of the sample superimposed with the spectrum of the 80 fs pump pulses used in the experiment, which were obtained from an amplifled CPM dye laser operating at 620 mn. Bottom absorption-difference spectra obtained at a series of probe time delays.

Messerschmidt, A., Landenstein, R., Huber, R., Bolognesi, M., Avigliano, L., Petruzzelli, R., Rossi, A. and Finazzi Agro, A. 1992. Refined crystal structure of ascorbate oxidase at 1.9 A resolution. Journal of Molecular Biology 224, 179-205. Miki, K., Ezoe, T., Masui, A., Yoshisaka, T., Mimuro, M., Fujiwara-Arasaki, T. and Kasai, N, 1990, Crystallization and preliminary X-ray diffraction studies of C-phycocyanin from a red alga, Porphyra tenera. Journal of Biochemistry 108, 646-649. Molecular Probes. Handbook of fluorescent probes and research chemicals. 1992-1994. 

PSII/PSI ratio 1500. The main pigments were phycocyanin (PC), allo-phycocyanin (AP) and chlorophyll (Chl). When excited at 445 nm the PSII preparations showed a minor fluorescence emission maximum at 660 nm characteristic for allophycocyanin and two major maxima at 685 and 693 nm The fluorescence at 685 nm was attributed to an interplay of allophycocyanin B, the large membrane-phycobilisome linker (Lem) and a Chl-antenna, whilst the 693 nm fluorescence belonged to a Chl-antenna alone. Most of the light energy captured by phycobilisomes was transferred to the final phycobilisome emitters and the PSII antennae as shown from the emission peak at 685 nm and the shoulder at 692 nm. 

Taken together. Figs. 1 and 2 indicate that levulinic acid interupts the recovery of cells from iron deficiency. We do not know if the inhibitor prevents iron uptake. Yet, it is clear that the biosynthesis and assembly of chlorophyll and phycocyanin into the membrane is depressed, yielding cells with enhanced original and depressed variable fluorescence. These characteristics might indicate a partial uncoupling of chlorophyll from PS II or PS I reaction centers, a conclusion which must await further testing. 

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