Pressurized Liquid Extraction of a Phycocyanobilin Chromophore and Its Reconstitution with a Cyanobacteriochrome Photosensor for Efficient Isotopic Labeling.

Linear tetrapyrrole compounds (bilins) are chromophores of the phytochrome and cyanobacteriochrome classes of photosensors and light-harvesting phycobiliproteins. Various spectroscopic techniques, such as resonance Raman, Fourier transform-infrared and nuclear magnetic resonance, have been used to elucidate the structures underlying their remarkable spectral diversity, in which the signals are experimentally assigned to specific structures using isotopically labeled bilin. However, current methods for isotopic labeling of bilins require specialized expertise, time-consuming procedures and/or expensive reagents. To address these shortcomings, we established a method for pressurized liquid extraction of phycocyanobilin (PCB) from the phycobiliprotein powder Lina Blue and also the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). PCB was efficiently cleaved in ethanol with three extractions (5 min each) under nitrogen at 125�C and 100 bars. A prewash at 75�C was effective for removing cellular pigments of Synechocystis without PCB cleavage. Liquid chromatography and mass spectrometry suggested that PCB was cleaved in the C3-E (majority) and C3-Z (partial) configurations. 15N- and 13C/15N-labeled PCBs were prepared from Synechocystis cells grown with NaH13CO3 and/or Na15NO3, the concentrations of which were optimized based on cell growth and pigmentation. Extracted PCB was reconstituted with a recombinant apoprotein of the cyanobacteriochrome-class photosensor RcaE. Yield of the photoactive holoprotein was improved by optimization of the expression conditions and cell disruption in the presence of Tween 20. Our method can be applied for the isotopic labeling of other PCB-binding proteins and for the commercial production of non-labeled PCB for food, cosmetic and medical applications.

Antioxidant activity of proteins extracted from red alga dulse harvested in Japan.

In this study, we investigated antioxidant activity of proteins from the red alga dulse (Palmaria sp.) harvested in Hokkaido, Japan. The dulse proteins that contain phycoerythrin (PE) as the main component showed a high radical scavenging activity. To clarify the key constituent of antioxidant activity in dulse proteins, we prepared recombinant dulse PE ß-subunit (rPEß) (apoprotein) and chromophores from the dulse proteins. As a result, the rPEß showed lower radical scavenging activity than that of dulse proteins. On the other hand, the dulse chromophores composed mainly of phycoerythrobilin (PEB) indicated extremely higher radical scavenging activity (90.4% ± 0.1%) than that of dulse proteins (17.9% ± 0.1%) on ABTS assay. In addition, on cell viability assay using human neuroblastoma SH-SY5Y cells, the dulse chromophores showed extracellular and intracellular cytoprotective effects against H2 O2 -induced cell damage. From these data, we concluded that the dulse proteins have antioxidant ability and the activity principally derives from the chromophores. PRACTICAL APPLICATION: Dulse is an abundant and underused resource, which contains a lot of proteins, especially phycoerythrin. We here demonstrated that the practically prepared dulse proteins possessed antioxidant activity and clarified that chromophores from the dulse proteins were the key components. Therefore, the dulse proteins have a potential for functional material.

Fast cleavage of phycocyanobilin from phycocyanin for use in food colouring.

Phycocyanins from cyanobacteria are possible sources for new natural blue colourants. Their chromophore, phycocyanobilin (PCB), was cleaved from the apoprotein by solvolysis in alcohols and alcoholic aqueous solutions. In all cases two PCB isomers were obtained, while different solvent adducts were formed upon the use of different reagents. The reaction is believed to take place via two competing pathways, a concerted E2 elimination and a SN2 nucleophilic substitution. Three cleavage methods were compared in terms of yield and purity: conventional reflux, sealed vessel heated in an oil bath, and microwave assisted reaction. The sealed vessel method is a new approach for fast cleavage of PCB from phycocyanin and gave at 120°C the same yield within 30min compared to 16h by the conventional reflux method (P<0.05). In addition the sealed vessel method resulted in improved purity compared to the other methods. Microwave irradiation increased product degradation.

On the Nature of Isomeric Nonfluorescent Chlorophyll Catabolites in Leaves and Fruit - A Study with a Ubiquitous Phylloleucobilin and its Main Isomerization Product.

The typical main products of chlorophyll (Chl) breakdown in higher plants are non-fluorescent, colorless phyllobilins, named phylloleucobilins. These long elusive Chl-catabolites are linear tetrapyrroles, whose structure elucidation has required thorough spectroscopic analyses. Interestingly, in recent LC/MS studies of leaf extracts, isomeric forms of phylloleucobilins were detected. The existence of isomeric phyllobilins may suggest incomplete stereo-selectivity of catabolic processes, or isomerization processes in plant cells or in the analytes. Here we report a study with the phylloleucobilin NCC-1, a basic Chl-catabolite in extracts of leaves and fruit. NCC-1 and its main isomerization product in aqueous solution were identified as 82 -epimers. Formation of 82 -epi-NCC-1 from NCC-1 implies an unstable enol(ate)-intermediate, which reverts to NCC-1 or converts to 82 -epi-NCC-1. Such reversible epimerization reactions are a non-biological in vitro feature of typical phylloleucobilins, and probably also take place in vivo.

Phycocyanobilin promotes PC12 cell survival and modulates immune and inflammatory genes and oxidative stress markers in acute cerebral hypoperfusion in rats.

Since the inflammatory response and oxidative stress are involved in the stroke cascade, we evaluated here the effects of Phycocyanobilin (PCB, the C-Phycocyanin linked tetrapyrrole) on PC12 cell survival, the gene expression and the oxidative status of hypoperfused rat brain. After the permanent bilateral common carotid arteries occlusion (BCCAo), the animals were treated with saline or PCB, taking samples 24h post-surgery. Global gene expression was analyzed with GeneChip Rat Gene ST 1.1 from Affymetrix; the expression of particular genes was assessed by the Fast SYBR Green RT-PCR Master Mix and Bioplex methods; and redox markers (MDA, PP, CAT, SOD) were evaluated spectrophotometrically. The PCB treatment prevented the H2O2 and glutamate induced PC12 cell injury assessed by the MTT assay, and modulated 190 genes (93 up- and 97 down-regulated) associated to several immunological and inflammatory processes in BCCAo rats. Furthermore, PCB positively modulated 19 genes mostly related to a detrimental pro-inflammatory environment and counteracted the oxidative imbalance in the treated BCCAo animals. Our results support the view of an effective influence of PCB on major inflammatory mediators in acute cerebral hypoperfusion. These results suggest that PCB has a potential to be a treatment for ischemic stroke for which further studies are needed.

Combinational biosynthesis of phycocyanobilin using genetically-engineered Escherichia coli.

Genes of the key enzymes for phycocyanobilin (PCB) biosynthesis were cloned into E. coli and combinationally expressed to produce phycocyanobilin, with autologous heme as substrate. Culture conditions were optimized to achieve ~3 mg PCB/l. A protocol for the purification of recombinant phycocyanobilin was established using solvent extraction combined with chromatography, which resulted in a final yield of ~0.3 mg PCB/l with a purity >95 %. Recombinant phycocyanobilin could scavenge hydroxyl radicals with an EC50 of 0.1 µM.

Phycocyanin and phycocyanobilin from Spirulina platensis protect against diabetic nephropathy by inhibiting oxidative stress.

We and other investigators have reported that bilirubin and its precursor biliverdin may have beneficial effects on diabetic vascular complications, including nephropathy, via its antioxidant effects. Here, we investigated whether phycocyanin derived from Spirulina platensis, a blue-green algae, and its chromophore phycocyanobilin, which has a chemical structure similar to that of biliverdin, protect against oxidative stress and renal dysfunction in db/db mice, a rodent model for Type 2 diabetes. Oral administration of phycocyanin (300 mg/kg) for 10 wk protected against albuminuria and renal mesangial expansion in db/db mice, and normalized tumor growth factor-ß and fibronectin expression. Phycocyanin also normalized urinary and renal oxidative stress markers and the expression of NAD(P)H oxidase components. Similar antioxidant effects were observed following oral administration of phycocyanobilin (15 mg/kg) for 2 wk. Phycocyanobilin, bilirubin, and biliverdin also inhibited NADPH dependent superoxide production in cultured renal mesangial cells. In conclusion, oral administration of phycocyanin and phycocyanobilin may offer a novel and feasible therapeutic approach for preventing diabetic nephropathy.

Clinical potential of phycocyanobilin for induction of T regulatory cells in the management of inflammatory disorders.

Exposure of human mononuclear cells to phycocyanin in vitro is reported to promote generation of Treg cells. Induction of heme oxygenase-1 (HO-1) in lymphocytes has a similar effect, and it is not likely to be accidental that a key product of HO-1 activity, biliverdin, is homologous to the structure of phycocyanin's chromophore phycocyanobilin (PhyCB). Moreover, Treg induction is observed in mice injected with bilirubin, biliverdin's chief metabolite. These considerations suggest that bilirubin, generated within lymphocytes by HO-1 activation, may play a physiological role in the promotion of Treg immunomodulation. This effect of bilirubin is likely to be independent of NADPH oxidase inhibition, since the NAPDH oxidase activity of macrophages is necessary for Treg induction, possibly because it contributes to HO-1 induction in lymphocytes. In light of numerous reports that oral phycocyanin is beneficial in various rodent models of autoimmune disorders, it is reasonable to suspect that PhyCB-enriched spirulina extracts may have clinical potential for boosting Treg activity in human autoimmune or allergic syndromes, mimicking the physiological role of HO-1 induction in this regard.

Assignment of phycocyanobilin in HMPT using triple resonance experiments.

A complete assignment of all resonances of a small organic molecule is a prerequisite for a structure determination using NMR spectroscopy. This is conventionally obtained using a well-established strategy based on COSY, HMQC and HMBC spectra. In case of phycocyanobilin (PCB) in HMPT this strategy was unsuccessful due to the symmetry of the molecule and extreme signal overlap. Since (13)C and (15)N labeled material was available, an alternative strategy for resonance assignment was used. Triple resonance experiments derived from experiments conventionally performed for proteins are sensitive and easy to analyze. Their application led to a complete and unambiguous assignment using three types of experiments.