Photoreaction in BLUF receptors: proton-coupled electron transfer in the flavin-Gln-Tyr system

Photoinduced electron transfer from tyrosine to the flavin chromophore is involved in activation of BLUF (sensor of blue light using FAD) photoreceptors. We studied the electron transfer (ET) coupled with proton-transfer (PT) reactions, by means of XMCQDPT2//CASSCF calculations on a molecular cluster model. By defining a minimum active space in the CASSCF calculations, we could compute the entire photoreaction pathway. We find that the crossing of the locally excited and ET states is located along the flavin bond-stretching coordinate. The ET state is stabilized by a proton transfer from the electron donor to the electron acceptor. We mapped two different PT pathways from tyrosine to flavin via the conserved glutamine. These reactions generate a tautomeric form of glutamine. Along the PT coordinates, we find geometries where the ET and the electronic ground states degenerate. At the state crossing structures, either formation of the ground state biradical intermediate or a relaxation back to the Franck-Condon minimum takes places. The computed relaxation pathways reveal that the hydrogen bonds involving glutamine in the chromophore-binding pocket control BLUF photoefficiency.     

Characterisation of mixed lithium dodecyl sulphate/lithium perfluorooctanesulphonate pseudo‐stationary phases in MEKC

Lipophilicity and methylene selectivity of mixed pseudo‐stationary phases (PSPs) (containing lithium dodecyl sulphate (LDS) and lithium perfluorooctanesulphonate (LiPFOS) in different molar ratios) applied in MEKC have been investigated. Micellar proportion (t_prop,mic, a quantity expressing that how much time is spent by the analyte in the micellar phase related to its whole migration time), CLOGP₅₀ value (showing the value of hydrophobicity of a molecule spending exactly 50% of its migration time in the PSP) and methylene selectivity have been determined for different LDS/LiPFOS mixed phases. Values of the above‐mentioned parameters have been determined for analytes with different chemical structures (alkylbenzene and alkylphenone homologous series, alcohols). Good linear correlation was obtained between either the micellar proportion, CLOGP₅₀, or methylene selectivity and the phase composition for the mixed phases. Lipophilicity and methylene selectivity of the mixed LDS/LiPFOS PSPs can be calculated and can continuously be changed by mixing the two single phases (LDS and LiPFOS) in the appropriate (and calculable) portion.     

Stripping voltammetry with the electrode material acting as a ‘built-in' internal standard

The oxidation peak of the bismuth layer of bismuth-film electrodes is used as an internal standard in anodic stripping voltammetry. Such use of the electrode material as a ‘built-in' internal standard simplifies the analytical protocol (as it obviates the need for an external standard or long calibration and standard additions procedures) while correcting for run-to-run variations due to uncertainties (e.g., changes in mass transport, surface area, or other variables). The new internal standard strategy relies on common in situ plated film electrodes, where the deposition of both the analyte and electrode material is subject to the same variations. Such one-step quantitation and improvements are illustrated for trace measurements of lead. The use of the intrinsic signal of the system for obtaining the concentration of the target analyte is unique not only for electroanalysis, but for analytical chemistry in general.     

Detection of tinuvin 770, a light stabilizer of plastic materials from dialysis membranes, by high-performance liquid chromatographic analysis

Tinuvin 770 [bis(2,2,6,6-tetramethyl-4-piperidinyl)sebacate] is a pharmacologically active agent used worldwide as a light stabilizer for plastic materials. In vitro studies show that it is an L-type Ca(2+) channel and neuronal nicotic acethylcholine receptor blocker. Hypotension, vegetative dysfunction, and neurological symptoms are frequently observed during a haemodialysis treatment. The release of Tinuvin 770 from plastic materials applied in haemodialysis may play a part in the development of clinical signs. In our study, four different commonly used haemodialysis membranes (polysulphon, cuprophan, and two types of hemophan) are examined. The polymers are soaked for 72 h in physiological saline solution. Isolation is carried out using a Waters Oasis SPE column for solid-phase extraction and by high-performance liquid chromatography (HPLC) with electrospray ionization-mass spectrometric detection. Tinuvin 770 release is detected from all examined membranes. Validation studies show a satisfactory selectivity, linearity, accuracy, and recovery of this method. Our results suggest that Tinuvin 770 could have specific toxicological and therapeutic importance related to haemodialysis treatment. The developed HPLC method is suitable for the detection of Tinuvin 770.     

Detection of a novel familial catalase mutation (Hungarian type D) and the possible risk of inherited catalase deficiency for diabetes mellitus

The enzyme catalase is the main regulator of hydrogen peroxide metabolism. Recent findings suggest that a low concentration of hydrogen peroxide may act as a messenger in some signalling pathways whereas high concentrations are toxic for many cells and cell components. Acatalasemia is a genetically heterogeneous condition with a worldwide distribution. Yet only two Japanese and three Hungarian syndrome-causing mutations have been reported. A large-scale (23 130 subjects) catalase screening program in Hungary yielded 12 hypocatalasemic families. The V family with four hypocatalasemics (60.6 +/- 7.6 MU/L) and six normocatalasemic (103.6 +/- 23.5 MU/L) members was examined to define the mutation causing the syndrome. Mutation screening yielded four novel polymorphisms. Of these, three intron sequence variations, namely G-->A at the nucleotide 60 position in intron 1, T-->A at position 11 in intron 2, and G-->T at position 31 in intron 12, are unlikely to be responsible for the decreased blood catalase activity. However, the novel G-->A mutation in exon 9 changes the essential amino acid Arg 354 to Cys 354 and may indeed be responsible for the decreased catalase activity. This inherited catalase deficiency, by inducing an increased hydrogen peroxide steady-state concentration in vivo, may be involved in the early manifestation of type 2 diabetes mellitus for the 35-year old proband.     

Ring[bond]chain tautomerism of 2-Aryl-substituted cis- and trans-decahydroquinazolines

In CDCl(3) at 300 K, 2-aryl-substituted cis- and trans-3-isopropyldecahydroquinazolines and trans-3-phenyldecahydroquinazolines proved to be three-component (r(1)[bond]o[bond]r(2)) ring[bond]chain tautomeric mixtures, whereas only ring-closed tautomers could be detected for the 3-methyl-substituted analogues. The proportions of the ring-chain tautomeric forms at equilibrium were strongly influenced by the N-substitutents and the cis-trans ring junction and could be described by the equation log K(X) = rho sigma(+) + log K(X=H). These are the first examples among 2-aryl-1,3-N,N-heterocycles of a three-component ring-chain tautomeric equilibrium characterized by a Hammett-type equation. The stabilities of the ring-closed forms of cis- and trans-2-aryldecahydroquinazolines and the corresponding 3,1-benzoxazines were found to increase in the following sequence of the heteroatom at position 3: NPh < N-i-Pr < O < NMe.     

Characterization of complex,heterogeneous lipid A samples using HPLC–MS/MS technique I. Overall analysis with respect to acylation,phosphorylation and isobaric distribution

We established a new reversed phase‐high performance liquid chromatography method combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry for the simultaneous determination and structural characterization of different lipid A types in bacteria (Escherichia coli O111, Salmonella adelaide O35 and Proteus morganii O34) showing serological cross‐reactivity. The complex lipid A mixtures (obtained by simple extraction and acid hydrolysis of the outer membrane lipopolysaccharides) were separated and detected without phosphate derivatization. Several previously unidentified ions were detected, which differed in the number and type of acyl chains and number of phosphate groups. In several cases, we observed the different retention of isobaric lipid A species, which had different secondary fatty acyl distribution at the C2′ or the C3′ sites. The fragmentation of the various, C4′ monophosphorylated lipid A species in deprotonated forms provided structural assignment for each component. Fragmentation pathways of the tri‐acylated, tetra‐acylated, penta‐acylated, hexa‐acylated and hepta‐acylated lipid A components and of the lipid A partial structures are suggested. As standards, the hexa‐acylated ion at m/z 1716 with the E. coli‐type acyl distribution and the hepta‐acylated ion at m/z 1954 with the Salmonella‐type acyl distribution were used. The results confirmed the presence of multiple forms of lipid A in all strains analyzed. In addition, the negative‐ion mode MS permitted efficient detection for non‐phosphorylated lipid A components, too. Copyright © 2016 John Wiley & Sons, Ltd.     

Conformational analysis of TMC114, a novel HIV-1 protease inhibitor

TMC114, a potent novel HIV-1 protease inhibitor, remains active against a broad spectrum of mutant viruses. In order to bind to a variety of mutants, the compound needs to make strong, preferably backbone, interactions and have enough conformational flexibility to adapt to the changing geometry of the active site. The conformational analysis of TMC114 in the gas phase yielded 43 conformers in which five types of intramolecular H-bond interactions could be observed. All 43 conformers were subject to both rigid and flexible ligand docking in the wild-type and a triple mutant (L63P/V82T/I84V) of HIV-1 protease. The largest binding energy was calculated for the conformations that are close to the conformation observed in the X-ray complexes of TMC114 and HIV-1 protease.