Characterization of the artificially covalent conjugate of B-phycoerythrin and R-phycocyanin and the phycobilisome from Porphyridium cruentum

B-phycoerythrin (BPE) and R-phycocyanin (RPC) were purified from Porphyridium cruentum by Sephadex G-200 chromatography, then the BPE was attached covalently to the RPC by reacting their amino groups to form the artificially covalent BPE-RPC conjugate in which the excitation energy can transfer from the BPE to the RPC with low efficiency. Meanwhile, the intact phycobilisome (PBS) consisting of BPE, RPC, APC and L-CM was isolated and purified from Porphyridium cruentum, and the purified PBS was found to keep intact if the solution contains sucrose. Comparison of spectroscopic properties between the purified PBS and the BPE-RPC conjugate suggests that the BPE-RPC conjugate is much more stable than the purified PBS. The construction of BPE-RPC conjugate with low efficiency of the excitation energy transfer may be useful for preparing phycobiliprotein probes. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Isolation, properties and spatial site analysis of gamma subunits of B-phycoerythrin and R-phycoerythrin

Polysiphonia urceolata R-phycoerythrin and Porphyridium cruentum B-phycoerythrin were degraded with proteinaseK, and then the nearly native gamma subunits were isolated from the reaction mixture. The process of degradation of phycoerythrin with proteinaseK showed that the gamma subunit is located in the central cavity of (alpha beta)(6) hexamer of phycoerythrin. Comparative analysis of the spectra of the native phycoerythrin, the phycoerythrin at pH 12 and the isolated gamma subunit showed that the absorption peaks of phycoerythrobilins on alpha or beta subunit are at 535 nm (or 545 nm) and 565 nm, the fluorescence emission maximum at 580 nm; the absorption peak of phycoerythrobilins on the isolated gamma subunit is at 589 nm, the fluorescence emission peak at 620 nm which overlaps the absorption maximum of C-phycocyanin and perhaps contributes to the energy transfer with high efficiency between phycoerythrin and phycocyanin in phycobilisome; the absorption maximum of phycourobilin on the isolated gamma subunit is at 498 nm, which is the same as that in native phycoerythrin, and the fluorescence emission maximum at 575 nm.

A novel vortex flow reactor for the purification of B-phycoerythrin from Porphyridium cruentum

The purification of B-phycoerythrin from a concentrated extract of disrupted Porphyridium cruentum cells was carried out using a new vortex flow reactor design for protein purification. The reactor behaved as an expanded bed in the laminar vortices flow regime where the Streamline DEAE resin was expanded by the axial flow and stabilized by the vortex flow. After the broth culture was centrifuged and resuspended in the adsorption buffer, the concentrated extract of disrupted cells was directly loaded into the vortex flow reactor. The purification of B-phycoerythrin was carried out in two steps: adsorption in the expanded bed and elution from the settled bed. 142.0 mg of B-phycoerythrin was eluted representing a total recovery yield of 86.6%. Prior to B-phycoerythrin purification, the protein adsorption of the vortex flow reactor was characterized through hydrodynamic studies and a dynamic capacity measurement using a standard protein.

The experimental research of R-phycoerythrin subunits on cancer treatment: A new photosensitizer in PDT

The mouse tumor cell 5180 and human liver carcinoma cell SMC 7721 cells were first treated with R-PE and its subunits (alpha, beta, gamma subunits), then irradiated with Argon laser (496 nm, 28.8 J/cm(2)). Survival rate was measured by MTT method. In order to compare the phototoxicity in normal cells, the mouse marrow cells were treated with photofrin II and beta-subunit, irradiated with 45 J/cm(2) of light; survival rate was also measured by MTT method. The result showed that R-PE subunits had better PDT effect on s180 cells than R-PE and lower phototoxicity in marrow cells than photofrin II Flow cytometric analysis showed that PDT results in a growth inhibition and a G(0)-G(1) cell cycle arrest in SMC 7721 cells. The tumor cells inhibited by PDT in vivo were morphologically observed by TEM, the tumor cell death was daze to the occlusion of tumor blood vessels and inducement of cell programmed death in nuclei. Therefore, with the advantage in special fluorescence activity, loth molecular weight, good light absorbent character and weak phototoxicity, R-PE subunit is art attractive option for improving the selectivity of PDT.

Proceso para la obtención y purificación de B-Ficoeritrina

Proceso en tres etapas para la obtención y purificación de la proteína B-ficoeritrina procedente de la microalga Porphyridium cruentum caracterizado por su alto rendimiento. La primera etapa consiste en una ruptura celular encaminada a liberar el material citoplasmático mediante un proceso de choque osmótico usando un tapón de ácido acético/acetato sódico. La segunda etapa utiliza un proceso cromatográfico en lecho expandido desarrollado en un columna de absorción rellena con un soporte absorbente iónico denominado Streamline-DEAE. Por último, la tercera etapa es un proceso adicional cromatográfico en columna de intercambio iónico de tipo clásico que utiliza como fase estacionaria un lecho de DEAE-celulosa DE-52.

Antioxidant activities of some tryptophan metabolites: possible implication for inflammatory diseases

The antioxidant properties of tryptophan and some of its oxidative metabolites were examined by measuring how efficiently they inhibited peroxyl radical-mediated oxidation of phosphatidylcholine liposomes and B-phycoerythrin. Low micromolar concentrations of 5-hydroxytryptophan, 3-hydroxykynurenine, xanthurenic acid, or 3-hydroxyanthranilic acid, but not their corresponding nonhydroxylated metabolic precursors, scavenged peroxyl radicals with high efficiency. In particular, 3-hydroxykynurenine and 3-hydroxyanthranilic acid protected B-phycoerythrin from peroxyl radical-mediated oxidative damage more effectively than equimolar amounts of either ascorbate or Trolox (a water-soluble analog of vitamin E). Enzyme activities involved or related to oxidative tryptophan metabolism, as well as endogenous concentrations of tryptophan and its metabolites, were determined within tissues of mice suffering from acute viral pneumonia. Infection resulted in a 100-fold induction of pulmonary indoleamine 2,3-dioxygenase (EC 1.13.11.17) as reported [Yoshida, R., Urade, Y., Tokuda, M. ; Hayaishi, O. (1979) Proc. Natl. Acad. Sci. USA 76, 4084-4086]. This was accompanied by a 16- and 3-fold increase in the levels of lung kynurenine and 3-hydroxykynurenine, respectively. In contrast, endogenous concentrations of tryptophan and xanthurenic acid did not increase and 3-hydroxyanthranilic acid could not be detected. The activity of the superoxide anion (O2-.)-producing enzyme xanthine oxidase increased 3.5-fold during infection while that of the O2-.-removing superoxide dismutase decreased to 50% of control levels. These results plus the known requirement of indoleamine 2,3-dioxygenase for superoxide anion for catalytic activity suggest that viral pneumonia is accompanied by oxidative stress and that induction of indoleamine 2,3-dioxygenase may represent a local antioxidant defence against this and possibly other types of inflammatory diseases.

Coexistence of phycoerythrin and a chlorophyll a/b antenna in a marine prokaryote.

Prochlorococcus marinus CCMP 1375, a ubiquitous and ecologically important marine prochlorophyte, was bound to possess functional genes coding for the alpha and beta subunits of a phycobiliprotein. The latter is similar to phycoerythrins (PE) from marine Synechococcus cyanobacteria and bind a phycourobilin-like pigment as the major chromophore. However, differences in the sequences of the alpha and beta chains compared with known PE subunits and the presence of a single bilin attachment site on the alpha subunit designate it as a novel PE type, which we propose naming PE-III. P. marinus is the sole prokaryotic organisms known so far that contains chlorophylls a and b as well as phycobilins. These data strongly suggest that the common ancestor of prochlorophytes and the Synechococcus cyanobacteria contained phycobilins. Flow cytometric data from the tropical Pacific Ocean provide evidence that deep populations of Prochlorococcus possess low amounts of a PE-like pigment, which could serve either in light harvesting or nitrogen storage or both.

Purification of R-phycoerythrin from Porphyra haitanensis (Bangiales, Rhodophyta) using expanded-bed absorption

R-phycoerythrin (R-PE) was purified from leafy gametophyte of Porphyra haitanensis T. J. Chang et B. F. Zheng (Bangiales, Rhodophyta) by a simple, scaleable procedure. Initially, phycobiliproteins were extracted by repeated freeze-thaw cycles, resulting in release from the algal cells by osmotic shock. Next, R-PE was recovered by applying the crude extract with a high concentration of (NH4)(2)SO4 salt directly to the expanded-bed columns loaded with phenyl-sepharose. An expanded-bed volume twice the settled-bed volume was maintained; then low (NH4)(2)SO4 concentration was used to develop the column. After two rounds of hydrophobic interaction chromatography (HIC), R-PE was purified by anion-exchange column. The method was also successful with free-living conchocelis of P. haitanensis. The purified R-PE was identified with electrophoresis, and absorption and fluorescence emission spectroscopy. The results were in agreement with those previously reported. The yield with a spectroscopic purity (OD565/OD280) higher than 3.2 (the ratio of A(565)/A(620) <= 0.02) was 1.4 mg . g(-1) of leafy gametophyte of P. haitanensis. For the free-living conchocelis of P. haitanensis extract, R-PE could be purified successfully with only one round of HIC. The yield with a spectroscopic purity (OD565/OD280) higher than 3.2 (the ratio of A(565)/A(620) <= 0.02) was 5.0 mg . g(-1) of free-living conchocelis of P. haitanensis. The method described here is a scaleable technology that allows a large quantity of R-PE to be recovered from the unclarified P. haitanensis crude extract. It is also a high protein recovery technology, reducing both processing costs and times, which enhances the value of this endemic Porphyra of China.

One-step chromatography method for efficient separation and purification of R-phycoerythrin from Polysiphonia urceolata

Phycoerythrins have been widely used in food, cosmetics., immunodiagnostics and analytical reagents. An efficient one-step chromatography method for purification of R-phycoerythrins from Polysiphonia urceolata was described in this paper. Pure R-phycoerythrin was obtained with an absorbance ratio A(565)/A(280) of 5.6 and a high recovery yield of 67-33%, using a DEAE-Sepharose Fast Flow chromatography with a gradient elution of pH, alternative to common gradient elution of ionic strength. The absorption spectrum of R-phycoerythrin was characterized with three absorbance maxima at 565, 539 and 498 mum, respectively and the fluorescence emission spectrum at room temperature was measured to be 580nm. The results of native-PAGE. and SDS-PAGE showed no contamination by other proteins in the phycoerythrin solution. which suggests an efficient method for the separation and purification of R-phycoerythrins from Polysiphonia urceolata. (C) 2004 Elsevier B.V. All rights reserved.

Phycoerythrin concentration during POLARSTERN cruises ANT-XXIV/4, ANT-XXV/1, and ANT-XXVI/4

Phycobiliproteins are a family of water-soluble pigment proteins that play an important role as accessory or antenna pigments and absorb in the green part of the light spectrum poorly used by chlorophyll a. The phycoerythrins (PEs) are one of four types of phycobiliproteins that are generally distinguished based on their absorption properties. As PEs are water soluble, they are generally not captured with conventional pigment analysis. Here we present a statistical model based on in situ measurements of three transatlantic cruises which allows us to derive relative PE concentration from standardized hyperspectral underwater radiance measurements (Lu). The model relies on Empirical Orthogonal Function (EOF) analysis of Lu spectra and, subsequently, a Generalized Linear Model with measured PE concentrations as the response variable and EOF loadings as predictor variables. The method is used to predict relative PE concentrations throughout the water column and to calculate integrated PE estimates based on those profiles.

Ultrasound-assisted extraction of R-phycoerythrin from Grateloupia turuturu with and without enzyme addition

The aim of this study was to compare two processes for the extraction of R-phycoerythrin (R-PE) from the red seaweed Grateloupia turuturu: ultrasound-assisted extraction (UAE) and ultrasound-assisted enzymatic hydrolysis (UAEH). Process efficiencies were both evaluated by the yield of R-PE extraction and by the level of liquefaction. Experiments were conducted at 40 and 22 °C, for 6 h, using an enzymatic cocktail and an original ultrasonic flow-through reactor. R-PE appeared very sensitive to temperature, thus 22 °C is strongly recommended for its extraction by UAEH or UAE. However, the higher processing temperature (40 °C) clearly increased the extraction of water-soluble compounds (up to 91% of liquefaction). These two new processes are thus promising alternatives for the extraction of water-soluble components including R-PE, from wet seaweeds, with extraction yields at least similar to conventional solid–liquid extraction.