A potent anti-oxidant property: fluorescent recombinant alpha-phycocyanin of Spirulina

To express and product a fluorescent antioxidant holo-alpha-phycocyanin (PC) of Spirulina platensis (Sp) with His-tag (rHHPC; recombinant holo-alpha-phycocyaninof Spirulina platensis with His-tag) in 5-l bench scale. A vector harbouring two cassettes was constructed: cpcA along with cpcE-cpcF in one cassette; ho1-pcyA in the other cassette. Lyases CpcE/F of Synechocystis sp. PCC6803 (S6) could catalyse the 82 site Cys in apo-alpha-PC of Sp linking with bilin chromophores, and rHHPC was biosynthesized in Escherichia coli BL21. The constant feeding mode was adopted, and transformant reached the biomass of rHHPC up to 0.55 g l(-1) broth in 5-litre bench scale. rHHPC was purified by Ni2+ affinity column conveniently. The absorbance and the fluorescence emission spectra of rHHPC had lambda(max) at 621 and 650 nm, respectively. The IC50 values of rHHPC were 277.5 +/- 25.8 mu g ml(-1) against hydroxyl radicals and 20.8 +/- 2.2 mu g ml(-1) against peroxyl radicals. Combinational biosynthesis of rHHPC was feasible, and the constant feeding mode was adopted to produce good yields of rHHPC. Fluorescent rHHPC with several unique qualitative and quantitative features was effective on scavenging hydroxyl and peroxyl radicals. A potent antioxidant rHHPC was co-expressed, produced and characterized for nutritional and pharmacological values, which would help to develop phycobiliproteins' applications in their fluorescent and biological activities.

Biosynthesis of fluorescent allophycocyanin alpha-subunits by autocatalysis in Escherichia coli

APC (allophycocyanin) is widely used for fluorescence tagging and may be a promising antioxidant agent for use within the food and pharmaceutical industries. Chromophore attachment to apo-ApcA (apo-APC alpha-subunit without chromophore) can be auto-catalysed both in vitro and in vivo. In the present study, a plasmid containing genes of apo-ApcA and chromophore synthetases (HOI (ferredoxin-dependent haem oxygenase) and PcyA (phycocyanobilin:ferredoxin oxidoreductase)] was constructed and expressed in Escherichia coli. The results show that holo-ApcA (APC alpha-subunit with chromophore) can be synthesized by autocatalysis in E. coli. Recombinant holo-ApcA showed the same spectral and fluorescent properties as PC (phycocyanin) and could serve as a good substitute for native PC for fluorescent tagging. Moreover, recombinant ApcA can inhibit hydroxyl and peroxyl radicals more strongly than holo-ApcA and native APC. The EC50 values were 296.4 +/- 22.4 mu g/ml against hydroxyl radicals and 38.5 +/- 2.6 mu g/ml against peroxyl radicals.

Combinational biosynthesis of a fluorescent cyanobacterial holo-alpha-allophycocyanin in Escherichia coli

Allophycocyanin ( APC) is a phycobiliprotein with various biological and pharmacological properties. An expression vector containing five essential genes in charge of biosynthesis of cyanobacterial APC holo-alpha subunit ( holo- ApcA) was constructed, resulting in over- expression of a fluorescent holo- ApcA in E. coli. After being cultured for 16 h, the dry cell density reached 22.5 gl(-1), and the expression of holo- HT- ApcA was up to 1 gl(-1) broth. The recombinant protein showed similar spectral features to native APC.

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC alpha and beta subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.

Authigenic sulfide minerals and their sulfur isotopes in sediments of the northern continental slope of the South China Sea and their implications for methane flux and gas hydrate formation

This is a report of the study of the authigenic sulfide minerals and their sulfur isotopes in a sediment core (NH-1) collected on the northern continental slope of the South China Sea, where other geophysical and geochemical evidence seems to suggest gas hydrate formation in the sediments. The study has led to the findings: (1) the pyrite content in sediments was relatively high and its grain size relatively large compared with that in normal pelagic or hemipelagic sediments; (2) the shallowest depth of the acid volatile sulfide (AVS) content maximum was at 437.5 cm (> 2 mu mol/g), which was deeper than that of the authigenic pyrite content maximum (at 141.5-380.5 cm); (3) delta S-34 of authigenic pyrite was positive (maximum: +15 parts per thousand) at depth interval of 250-380 cm; (4) the positive delta S-34 coincided with pyrite enrichment. Compared with the results obtained from the Black Sea sediments by Jorgensen and coworkers, these observations indicated that at the NH-1 site, the depth of the sulfate-methane interface (SMI) would be or once was at about 437.5-547.5 cm and the relatively shallow SMI depth suggested high upward methane fluxes. This was in good agreement with the results obtained from pore water sulfate gradients and core head-space methane concentrations in sediment cores collected in the area. All available evidence suggested that methane gas hydrate formation may exist or may have existed in the underlying sediments.

Characteristics of clay minerals in the northern South China Sea and its implications for evolution of East Asian monsoon since Miocene

Clay mineral assemblages, crystallinity, chemistry, and micromorphology of clay particles in sediments from ODP Site 1146 in the northern South China Sea (SCS) were analyzed, and used to trace sediment sources and obtain proxy records of the past changes in the East Asian monsoon climate since the Miocene, based on a multi-approach, including X-ray diffraction (XRD) and scanning electron microscopy combined with energy dispersive X-ray spectrometry (SEM-EDS). Clay minerals consist mainly of illite and smectite, with associated chlorite and kaolinite. The illite at ODP Site 1146 has very well-to-well crystallinity, and smectite has moderate-to-poor crystallinity. In SEM the smectite particles at ODP Site 1146 often appear cauliflower-like, a typical micromorphology of volcanic smecites. The smectite at ODP Site 1146 is relatively rich in Si element, but poor in Fe, very similar to the smectite from the West Philippine Sea. In contrast, the chemical composition of illite at ODP Site 1146 has no obvious differences from those of the Loess plateau, Yellow River, Yangtze River, and Pearl River. A further study on sediment source indicates that smectite originates mainly from Luzon, kaolinite from the Pearl River, and illite and chlorite from the Pearl River, Taiwan and/or the Yangtze River. The clay mineral assemblages at ODP Site 1146 were not only controlled by continental eathering regimes surrounding the SCS, but also by the changing strength of the transport processes. The ratios of (illite+chlorite)/smectite at ODP Site 1146 were adopted as proxies for the East Asian monsoon evolution. Relatively higher ratios reflect strongly intensified winter monsoon relative to summer monsoon, in contrast, lower ratios indicate a strengthened summer monsoon relative to winter monsoon. The consistent variation of this clay proxy from those of Loess plateau, eolian deposition in the North Pacific, planktonic, benthic foraminifera, and black carbon in the SCS since 20 Ma shows that three profound shifts of the East Asian winter monsoon intensity, and aridity in the Asian inland and the intensity of winter monsoon relative to summer monsoon, occurred at about 15 Ma, 8 Ma, and the younger at about 3 Ma. The phased uplift of the Himalaya-Tibetan plateau may have played a significant role in strengthening the Asian monsoon at 15 Ma, 8 Ma, and 3 Ma.

Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry

A pre-column derivatization method for the sensitive determination of amino acids and peptides using the tagging reagent 1,2-benzo-3,4dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS/MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEOC. BCEOC can easily and quickly label peptides and amino acids. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z (M + H)(+) under electrospray ionization (ESI) positive-ion mode with an exception being Tyr detected at negative mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 246.2 corresponding to the cleavage of C-O bond of BCEOC molecule. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% are observed with a 3-4-fold molar reagent excess. Derivatives exhibit strong fluorescence and extracted detzvatization solution with n-hexane/ethyl acetate (10:1, v/v) allows for the direct injection with no significant interference from the major fluorescent reagent degradation by-products, such as 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDC-OH) (a major by-product), mono- 1,2-benzo-3,4-dihydrocarbazole-9-ethyl carbonate (BCEOC-OH) and bis-(1,2-benzo-3,4-dihydrocarbazole-9-ethyl) carbonate (BCEOC)(2). In addition, the detection responses for BCEOC derivatives are compared to those obtained with previously synthesized 2-(9-carbazole)-ethyl chloroformate (CEOC) in our laboratory. The ratios AC(BCEOC)/AC(CEOC) = 2.05-6.51 for fluorescence responses are observed (here, AC is relative fluorescence response). Separation of the derivatized peptides and amino acids had been optimized on Hypersil BDS C-18 column. Detection limits were calculated from 1.0 pmol injection at a signal-to-noise ratio of 3, and were 6.3 (Lys)-177.6 (His) fmol. The mean interday accuracy ranged from 92 to 106% for fluorescence detection with mean %CV < 7.5. The mean interday precision for all standards was < 10% of the expected concentration. Excellent linear responses were observed with coefficients of > 0.9999. Good compositional data could be obtained from the analysis of derivatized protein hydrolysates containing as little as 50.5 ng of sample. Therefore, the facile BCEOC derivatization coupled with mass spectrometry allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids and peptides from biological and natural environmental samples. (c) 2005 Elsevier B.V. All rights reserved.

Synthesis of a terbium fluorescent chelate and its application to time-resolved fluoroimmunoassay

A new nonadentate ligand, N, N, N-1, N-1-[2,6-bis(3'-aminomethyl-1 1'-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA) for a Tb3+ fluorescent complex was synthesized. The Tb3+ complex is strongly fluorescent, having a large fluorescence quantum yield of 1.00 and very long fluorescence lifetime of 2.681 ms in 0.05 M berate buffer of pH 9.1. Streptavidin (SA) was labeled with SPTA by using its succinimidyl monoester, and the BPTA-Tb3+-labeled SA was used in sandwich-type time-resolved fluoroimmunoassay (TR-FIA) of alpha -fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera. The Tb3+-labeled SA was also used in competitive type TR-FIA of bensulfuron- methyl (BSM) in water. The detection limits of these assays are 42 pg/mL for AFP, 70 pg/mL for CEA, and 0.4 ng/mL for BSM. In addition, a new simultaneous measurement method for AFP and CEA in a human serum sample was developed by using 4,4'-bis(1 " ,1 " ,1 " ,2 " ,2 " ,3 " ,3 " -heptafluoro-4 " ,6 " -hexanedion-6 " -yl)chlorosulfo-o-terphenyl ((BHHCT)-Eu3+-labeled anti-AFP antibody, biotinylated anti-CEA antibody, and BPTA-Tb3+-labeled SA. The concentrations of AFP and CEA in 39 human serum samples were determined, and the results were compared with those of the independently determined AFP and CEA by TR-FIA with a single-label method. A good correlation was obtained with the correlation coefficients of 0.991 for AFP and 0.994 for CEA.

Preparation and a time-resolved fluoroimmunoassay application of new europium fluorescent nanoparticles

New silica-based europium fluorescent nanoparticles having surface amino groups were prepared by a covalent binding-copolymerization technique. In the nanoparticles, the fluorescent Eu3+ chelate molecules were covalently bound to silicon atoms to protect the nanoparticles from dye leaking in bio-applications. The amino groups on the surface of nanoparticles made the surface modification and bioconjugation of nanoparticles easier. The nanoparticles were characterized and developed as a new type of fluorescence probe for a highly sensitive time-resolved fluoroimmunoassay (TR-FIA) of human hepatitis B surface antigen (HBsAg).