Phycobiliprotein

FLUORESCENT PINK FROM RED PORPHYRIDIUM MICROALGA AND PHYCOBILIPROTEINS... 

The red microalga genus Porphyridium is a source of biochemicals possessing nutritional and therapeutic values. The biochemicals include polysaccharides that have anti-inflammatory and antiviral properties, long-chain polyunsaturated fatty acids, carotenoids such as zeaxanthin, and fluorescent phycobiliproteins. 

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 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. 

Yaron, A. and Arad (Mails), S., Phycobiliproteins — blue and red natural pigments — for use in food and cosmetics, in Food Flavors, Ingredients and Composition Developments in Food Science, Vol. 32, Charalambous, G., Ed., Elsevier, London, 1993, 835. 

The number of new NIR fluorophores that can be used in biological systems has grown substantially in recent years as a consequence of extensive research efforts to improve the properties of available dyes. A brief overview of the various types of long-wavelength (above 600 nm) fluorophores including phycobiliproteins, BODIPY, and Alexa Fluor dyes (Life Technologies), Cy dyes (GE Healthcare),... 

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. 

As mentioned above, in all photophobic action spectra measured so far additional peaks and/or shoulders occur at 565 and/or 615 nm, besides the chlorophyll a peaks, indicating the participation of phycobiliproteins and, hence, PS II in the active light absorption (Fig. 4). Moreover, activity of both PS I and PS II was confirmed by inhibitor experiments (see reviews by Nultsch 10°),Nultsch and Hader102), and Hader47)) and also with the aid of a dual wavelength technique39 40 101), described in detail below. 

These experiments show that, as in the case of chlorophyll a and the carotenoids, the energy absorbed by the phycobiliproteins is utilized via the photosynthetic apparatus furthermore, they provide evidence that photophobic responses in blue-green algae are caused by sudden changes in the steady state of the photosynthetic electron transport, especially the non-cyclic one. 

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,... 

Phycobiliprotein Molecular weight Absorption maximum (nm) EC (cm-1 M 1) Emission maximum (nm) Fluorescence QY... 

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). 

Preparation of a series of phycobiliprotein tandem dyes allows multiplexed analysis of different targets in a sample. In addition, since RPE can be excited by the argon-ion laser at 488 nm, a fluorescein-labeled probe can be used concurrently with RPE alone and RPE-tandem conjugates to create a multiplexed system of different fluorescent probes that can be used simultaneously. Table 9.3 shows the different combinations of dyes that can be used in this type of assay with RPE and APC. 

Dialyze the phycobiliprotein into 50mM sodium borate, 0.3M NaCl, pH 8.5 (Note Commercial preparations of these proteins come as an ammonium sulfate suspension). 

The SPDP-activated phycobiliprotein may be reacted with a sulfhydryl-containing protein to create a fluorescent conjugate linked through disulfide bonds. 

Other proteins commonly crosslinked to (strept)avidin are chromogenic or fluorescent molecules, such as ferritin or phycobiliproteins (Chapter 9, Section 7). These conjugates can be used in microscopy techniques to stain and localize certain antigens or receptors in cells or tissue sections. 

Conjugates of (strept)avidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to create a sulf-hydryl-reactive derivative, followed by modification of (strept)avidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of (strept)avidin can be found in Section 3.1, this chapter. The procedure for SPDP activation of phycobiliproteins can be found in Chapter 9, Section 7. Reacting the SPDP-activated phycobiliprotein with thiol-labeled (strept)avidin at a molar ratio of 2 1 will result in highly fluorescent biotin binding probes. 

Glazer, A.N., and Stryer, L. (1983) Fluorescent tandem phycobiliprotein conjugates Emission wavelength shifting by energy transfer. Biophys. /. 43, 383-386. 

Grabowski, J., and Gantt, E. (1978) Photophysical properties of phycobiliproteins from phycobilisomes Fluorescence lifetimes, quantum yields, and polarization spectra. Photochem. Pbotobiol. 28, 39-45. 

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