Differential potentiometric stripping analysis

Differential potentiometric stripping analysis, a sensitive instrumental modification of potentiometric stripping analysis, is described. For trace elements like cadmium and lead, which exhibit transport-controlled potentiometric stripping, signal enhancement is possible by employing a scheme involving multiple stripping and re-reduction of the preconcentrated analytes. For such elements the detection limit is below 5 × 10^-10 M with 60-s plating. The accuracy of the technique is tested on a biological reference material. Like potentiometric stripping analysis, the technique presented is not sensitive to reversible redox couples in solution.     

De novo metallonucleases based on helix-loop-helix motifs

Three new 42-mer peptides (PR I-III) designed to fold into a hairpin helix-loop-helix motif have been prepared. In the peptide sequence two (PR II-III) or four (PR I) copies of an unnatural amino acid bearing a triazacyclononane metal-ion binding site (ATANP) have been inserted in appropriate positions to allow the ligand subunits to face each other either within the same helix or between the two helices of the hairpin motif. Circular dichroism (CD) studies in solution have shown that the apopeptides adopt a well-defined helix-loop-helix tertiary structure that dimerizes in solution at concentrations above 200 microM to form a four-helix bundle. However, the helical content is strongly dependent on pH and metal-ion binding. Both protonation of the amines of the triazacyclononane units present in the ATANP lateral arm and complexation with Zn(II) ions cause a significant decrease of the helical content of the sequences. The Zn(II) complexes of the three peptides catalyze the transesterification of the RNA model substrate 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP) with different efficiency. The best catalyst appears to be PR I-4 Zn(II), that is, the peptide incorporating four ATANP units. Michaelis-Menten saturation kinetics allowed us to estimate that substrate fully bound to the catalyst reacts 380 times faster than in its absence. The kinetic evidence suggests cooperativity between (at least two) metal ions: one activating the nucleophilic species (directly or indirectly) and the other facilitating nucleophilic attack by coordination of the phosphate.     

Methyl 4‐O‐benzoyl‐2,3‐O‐isopropyl­idene‐α‐l‐rhamnopyran­oside

The title compound, C₁₇H₂₂O₆, has an exocyclic ester group at the hexopyranosyl sugar residue. The carbonyl group shows a conformation that is eclipsed with respect to the adjacent ring C—H bond. The two ester torsion angles are denoted by syn and cis conformations. One of these torsion angles is indicated to have a similar conformation in solution, as analyzed by NMR spectroscopy and a Karplus‐type relationship.     

LIGHT-DRIVEN H2 PRODUCTION WITH PROFLAVIN AND HYDROGENASE: COMPARISON OF CYTOCHROME C3 AND METHYL VIOLOGEN AS e- MEDIATORS

We have studied the hydrogenase-catalyzed production of H, when either its natural electron mediator cytochrome c₃ (c₃) or the artificial mediator methyl viologen (MV) was reduced by illumination of proflavin (PF) in the presence of ethylenediaminetetraacetate (EDTA). The reduction rates of MV and c₃ were comparable at equivalent concentrations of PF, taking into account the four redox sites of c₃. However, when the concentration of c₃ exceeded that of PF, the reduction rate decreased. We explain this by light quenching. In the H₂-producing system, MV was more efficient than c₃ as electron mediator when the initial reaction rates were compared. However, under certain conditions with MV, the rate of H₂ production decreased rapidly with time of illumination, whereas with c₃ it consistently remained stable. Possible reasons for this difference are discussed.
Severe inactivation of hydrogenase was observed in the absence of the primary electron donor EDTA. It is concluded that this inactivation is caused by the excited state of PF     

UV-induced changes in pigment content and light penetration in the fruticose lichen Cladonia arbuscula ssp. mitis

The response of the lichen, Cladonia arbuscula (Wallr.) Flot. ssp. mitis (Sandst.) Ruoss to enhanced UV-B (280-315 nm) radiation was investigated with respect to: (a) changes in phenolic content; (b) differential pigment accumulation under visible and UV radiation with increasing distance from thallus apices; and (c) the internal distribution of UV-B radiation within the thallus measured with quartz optical fibres. In a short-term experiment, lichens were exposed for 7 days in a growth chamber to visible light with or without additional UV-B radiation. For a longer term experiment, lichens were grown outdoors under both natural UV radiation, and supplemental UV-A (315-400 nm)+UV-B provided by lamps. Controls were placed under filters that removed the radiation below 290 nm from the natural sunlight. The concentration of total phenolic compounds was measured spectrophotometrically at the termination of the experiments, in different parts of the lichen podetia. UV-exposed lichens showed increased accumulation of phenolics compared to those not grown under UV. At the termination of the long-term experiment, fibre optic measurements of the penetration of radiation into lichen thallus reflected the influence of growth under UV radiation, whereby UV was more strongly attenuated as compared to that in lichens not exposed to enhanced levels of UV-B radiation. Results indicated that in Cladonia, UV-B radiation induces the accumulation of phenolic compounds that may have a protective role. In addition, the morphological distribution of phenolic compounds was different under visible and supplemental UV-B radiation. Internal radiation measurements served to visualise the attenuation of radiation with thallus depth for different wavelengths in the UV-B waveband.     

Flow potentiometric and constant-current stripping analysis for silver(I) with carbon- and platinum-fibre electrodes

At concentrations above 50 μg l^?1, silver(I) is determined in nitric acid medium by means of potentiostatic deposition onto a platinum-fibre electrode and subsequent constant-current stripping in the sample or potentiometric stripping in a potassium permanganate medium. Interference from copper(II) is reduced by a pulsed potential procedure whereby copper deposited onto the fibre electrode is reoxidized intermittently. At concentrations below 50 μg l^?1, silver(I) is determined by using a mercury-coated carbon-fibre electrode and constant-current stripping in acetonitrile containing 0.20 M perchloric acid. Potentiostatic deposition for 30 min yielded a detection limit of 0.24 μg l^?1 silver(I) at the 3σ level.     

TiCl4 Dissolved in Ionic Liquid Mixtures from Аb Initio Molecular Dynamics Simulations

To gain a deeper understanding of the TiCl4 solvation effects in multi-component ionic liquids, we performed ab initio molecular dynamics simulations of 1-butyl-3-methylimidazolium [C4C1Im]+, tetrafluoroborate [BF4]−, chloride [Cl]− both with and without water and titanium tetrachloride TiCl4. Complex interactions between cations and anions are observed in all investigated systems. By further addition of water and TiCl4 this complex interaction network is extended. Observations of the radial distribution functions and number integrals show that water and TiCl4 not only compete with each other to interact mainly with [Cl]−, which strongly influences the cation-[BF4]− interaction, but also interact with each other, which leads to the fact that in certain systems the cation-anion interaction is enhanced. Further investigations of the Voronoi polyhedra analysis have demonstrated that water has a greater impact on the nanosegregated system than TiCl4 which is also due to the fact of the shear amount of water relative to all other components and its higher mobility compared to TiCl4. Overall, the polar network of the IL mixture collapses by including water and TiCl4. In the case of [Cl]− chloride enters the water continuum, while [BF4]− remains largely unaffected, which deeply affects the interaction of the ionic liquid (IL) network.     

Quantum mechanical/molecular mechanical free energy simulations of the glutathione S-transferase (M1-1) reaction with phenanthrene 9,10-oxide

Glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotics in mammals. They catalyze the conjugation of glutathione to a wide range of electrophilic compounds. Phenanthrene 9,10-oxide is a model substrate for GSTs, representing an important group of epoxide substrates. In the present study, combined quantum mechanical/molecular mechanical (QM/MM) simulations of the conjugation of glutathione to phenanthrene 9,10-oxide, catalyzed by the M1-1 isoenzyme from rat, have been carried out to obtain insight into details of the reaction mechanism and the role of solvent present in the highly solvent accessible active site. Reaction-specific AM1 parameters for sulfur have been developed to obtain an accurate modeling of the reaction, and QM/MM solvent interactions in the model have been calibrated. Free energy profiles for the formation of two diastereomeric products were obtained from molecular dynamics simulations of the enzyme, using umbrella sampling and weighted histogram analysis techniques. The barriers (20 kcal/mol) are in good agreement with the overall experimental rate constant and with the formation of equal amounts of the two diastereomeric products, as experimentally observed. Along the reaction pathway, desolvation of the thiolate sulfur of glutathione is observed, in agreement with solvent isotope experiments, as well as increased solvation of the epoxide oxygen of phenanthrene 9,10-oxide, illustrating an important stabilizing role for active site solvent molecules. Important active site interactions have been identified and analyzed. The catalytic effect of Tyr115 through a direct hydrogen bond with the epoxide oxygen of the substrate, which was proposed on the basis of the crystal structure of the (9S,10S) product complex, is supported by the simulations. The indirect interaction through a mediating water molecule, observed in the crystal structure of the (9R,10R) product complex, cannot be confirmed to play a role in the conjugation step. A selection of mutations is modeled. The Asn8Asp mutation, representing one of the differences between the M1-1 and M2-2 isoenzymes, is identified as a possible factor contributing to the difference in the ratio of product formation by these two isoenzymes. The QM/MM reaction pathway simulations provide new and detailed insight into the reaction mechanism of this important class of detoxifying enzymes and illustrate the potential of QM/MM modeling to complement experimental data on enzyme reaction mechanisms.     

Effects of pH on the kinetic reaction

In most cases, kinetic unfolding reactions of proteins follow a simple one-step mechanism that does not involve any detectable intermediates. One example for a more complicated unfolding reaction is the acid-induced denaturation of holo-myoglobin (hMb). This reaction proceeds through a transient intermediate and can be described by a sequential two-step mechanism (Konermann et al. Biochemistry 1997, 36, 6448-6454). Time-resolved electrospray ionization mass spectrometry (ESI MS) is a new technique for monitoring the kinetics of protein folding and unfolding in solution. Different protein conformations can be distinguished by the different charge state distributions that they generate during ESI. At the same time this technique allows monitoring the loss or binding of noncovalent protein ligands. In this work, time-resolved ESI MS is used to study the dependence of the kinetic unfolding mechanism of hMb on the specific solvent conditions used in the experiment. It is shown that hMb unfolds through a short-lived intermediate only at acidic pH. Under basic conditions no intermediate is observed. These findings are confirmed by the results of optical stopped-flow absorption experiments. This appears to be the first time that a dependence of the kinetic mechanism for protein unfolding on external conditions such as pH has been observed.