Q95484844
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Reliable analysis of anions in mine waters with high iron and sulfate contents is important for their characterization. However, both traditional and LC methods failed in direct anion analysis. The article describes two sample pretreatments – alkalization and cation exchange. The pretreated water samples were analyzed by potentiometric titration, spectrophotometry, ion chromatography with conductivity detector in suppressed regime and HPLC with diode array detector using indirect UV detection at 260 nm. Both the sample pretreatments enable determination of some anions, the former procedure being more suitable for the purpose. Potentiometric titration and spectrophotometry seem to be the most suitable due to low detection limits for all the selected anions ranging from 0.4 to 1.8 mg l–1. The application of ion chromatography and liquid chromatography with diode array detector for the purpose is also possible. However, due to very high detection limits of samples pretreated by cation exchange (26–121 mg l–1) and low recoveries of samples pretreated by alkalization (<17 %) for all the selected anions, HPLC-DAD is not suitable for analysis of real samples. Validation and analytical characteristics of the methods are given and discussed.
BACKGROUND: The Nse1, Nse3 and Nse4 proteins form a tight sub-complex of the large SMC5-6 protein complex. hNSE3/MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and the Nse4 kleisin subunit is related to the EID (E1A-like inhibitor of differentiation) family of proteins. We have recently shown that human MAGE proteins can interact with NSE4/EID proteins through their characteristic conserved hydrophobic pocket. METHODOLOGY/PRINCIPAL FINDINGS: Using mutagenesis and protein-protein interaction analyses, we have identified a new Nse3/MAGE-binding domain (NMBD) of the Nse4/EID proteins. This short domain is located next to the Nse4 N-terminal kleisin motif and is conserved in all NSE4/EID proteins. The central amino acid residues of the human NSE4b/EID3 domain were essential for its binding to hNSE3/MAGEG1 in yeast two-hybrid assays suggesting they form the core of the binding domain. PEPSCAN ELISA measurements of the MAGEC2 binding affinity to EID2 mutant peptides showed that similar core residues contribute to the EID2-MAGEC2 interaction. In addition, the N-terminal extension of the EID2 binding domain took part in the EID2-MAGEC2 interaction. Finally, docking and molecular dynamic simulations enabled us to generate a structure model for EID2-MAGEC2. Combination of our experimental data and the structure modeling showed how the core helical region of the NSE4/EID domain binds into the conserved pocket characteristic of the MAGE protein family. CONCLUSIONS/SIGNIFICANCE: We have identified a new Nse4/EID conserved domain and characterized its binding to Nse3/MAGE proteins. The conservation and binding of the interacting surfaces suggest tight co-evolution of both Nse4/EID and Nse3/MAGE protein families.
- MeSH
- interakční proteinové domény a motivy MeSH
- intracelulární signální peptidy a proteiny chemie genetika metabolismus MeSH
- jaderné proteiny chemie MeSH
- konzervovaná sekvence MeSH
- lidé MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- mutageneze cílená MeSH
- peptidové fragmenty chemie genetika metabolismus MeSH
- počítačová simulace MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- Schizosaccharomyces pombe - proteiny chemie MeSH
- Schizosaccharomyces MeSH
- sekvence aminokyselin MeSH
- substituce aminokyselin MeSH
- techniky dvojhybridového systému MeSH
- transportní proteiny chemie genetika metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To fulfil the requirements of health regulations and water quality, it is necessary to monitor the concentrations of volatile hydrocarbons in water at rather low levels. A simple and rapid GC-FID method utilizing liquid-liquid microextraction with xylene in closed vials for the determination of ten selected volatile chlorinated hydrocarbons in water before and after remediation was developed. The procedure allowed to determine selected hydrocarbons at concentrations 10?20 µg l-1 using 1000-ml samples or 30?50 µg l-1 using 60-ml samples. A significant decrease in the detection limits to 2?20 µg l-1 (for 60-ml samples) was achieved by lowering the noise level after replacing GC gases by higher-purity gases and using 1-chloropropane as internal standard. The determination of CHCl3 and CCl4 at the µg l-1 levels was feasible using an ECD detector. Even lower detection limits can be achieved by modification of the GC injector and by injecting a large volume of the xylene extract.
