B-phycoerythrin

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. 

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. 

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,... 

R. Bermejo, E.M. Talavera and J.M. Alvarez-Pez, Chromatographic purification and characterization of B-phycoerythrin of Porphyridium cruentum. Semipreparative high-performance liquid chromatographic separation and characterization of its subunits. J. Chromatogr.A 917 (2001) 135-145. 

B-phycoerythrin-labeled antibody (BPE-IgG), and that use Texas Red-labeled antibody as an acceptor for BPE-IgG.(24)... 

Abbreviations AMCA, 7-amino-4-methylcoumarin B-PE, B phycoerythrin Cy, cyanine DAMC, diethylaminocoumarin FITC, fluorescein isothiocyanate RB-200-SC, lissamine rhodamine sulfonylchloride R-PE, R phycoerythrin SITS, 4-acetamido-4 -isothiocyanato-stilbene-2,2 -disulfonic acid TRITC, tetramethyl rhodamine isothiocyanate XRITC, rhodamine X isothiocyanate. Information obtained from refs. 2, 9, and 10. 

Oxygen radical absorbency capacity (ORAC) method presumes registration of substrate (B-phycoerythrin or fluorescein) fluorescence [43,44] after reaction with AOC in the sample to be compared with reference template. Trolox is used as a standard to determine per-oxyl radicals and gallic acid to determine hydroxyl radicals. 

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... 

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.

Abbreviations used FITC = fluorescein isothiocyanate DTAF = dichloro-triazinylaminofluorescein, TRITC = tetramethylrhodamine isothiocyanate, RB-200SC s lissamine rhodamine sulfonylchloride, RBITC = rhodamine B isothiocyanate, XRITC = rhodamine X isothiocyanate CY3.18 = cyanine 3.18, B-PE = B phycoerythrin, R-PE = R phycoerythrin SITS = 4-acetamido-4 -isothiocyanatostilbene-2,2 -disulfonic acid, DAMC = diethylaminocoumarin, AMCA = 7-Amino-4-methylcoumarin-3-acetic acid. Information obtained from refs. (2,9,10)... 

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. 

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.

Fig. S. Spatial model for antibody-antigen complexes involving B-phycoerythrin (B-PE) as energy acceptor and antibodies to human immunoglobulin G loaded with the energy donor pyrene butyrate. The shaded circles represent fluorescent antibody fragments covalently labeled with B-PE. (From Morrison. ...

In this section, we provide some details on the detection of individual molecules of B-phycoerythrin, separated by capillary electrophoresis. B-phycoerythrin is a large protein found in the light harvesting apparatus of certain algae. The protein exists in several different forms, which reflect different decorations added by nature to the peptide backbone of the molecule. However, before discussing the capillary electrophoresis results, we describe the performance of the detector. 

We relied on electrokinetic pumping to introduce analyte molecules into the cuvette at very low rates. The injection tip of the capillary was immersed in a small reservoir that contained B-phycoerythrin at a concentration of 1.7 x 10 mol 1 . A platinum electrode was also immersed in the sample, and a several kilovolt potential was applied to the electrode. It is important to encase the electrode in an interlock equipped enclosure to prevent accidental contact with the high voltage. 

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