Binding of single nucleotides to H+-ATP synthases observed by fluorescence resonance energy transfer

F(0)F(1)-ATP synthases couple proton translocation with the synthesis of ATP from ADP and phosphate. The enzyme has three catalytic nucleotide binding sites, one on each beta-subunit; three non-catalytic binding sites are located mainly on each alpha-subunit. In order to observe substrate binding to the enzyme, the H(+)-ATP synthase from Escherichia coli was labelled selectively with the fluorescence donor tetramethylrhodamine (TMR) at position T106C of the gamma-subunit. The labelled enzymes were incorporated into liposomes and catalysed proton-driven ATP synthesis. The substrate ATP-Alexa Fluor 647 was used as the fluorescence acceptor to perform intermolecular fluorescence resonance energy transfer (FRET). Single molecules are detected with a confocal set-up. When one ATP-Alexa Fluor 647 binds to the enzyme, FRET can be observed. Five stable states with different intermolecular FRET efficiencies were distinguished for enzyme-bound ATP-Alexa Fluor 647 indicating binding to different binding sites. Consecutive hydrolysis of excess ATP resulted in stepwise changes of the FRET efficiency. Thereby, gamma-subunit movement during catalysis was directly monitored with respect to the binding site with bound ATP-Alexa Fluor 647.     

Atomic layer deposition in nanometer-level replication of cellulosic substances and preparation of photocatalytic TiO2/cellulose composites

TiO2 replicas of filter paper with nanometer-level accuracy were prepared by atomic layer deposition of thin conformal TiO2 coating, followed by a removal of the paper by air-anneal at 450 degrees C. Photocatalytic anatase TiO2/cellulose composites were also made by leaving the paper intact. The TiO2 films were deposited from Ti(OMe)4 and H2O at 150-250 degrees C. The photocatalytic activity of the TiO2/cellulose composite was verified by photocatalytic reduction of Ag(I) from an aqueous solution to Ag nanoparticles on the TiO2 surface. The TiO2/cellulose composites are mechanically more stable than the free-standing TiO2 replicas and are therefore potentially suitable as lightweight, high surface area photocatalysts.     

Speciation in the aqueous H+/H2VO4-/H2O2/picolinate system relevant to diabetes research

A detailed study of the quaternary aqueous H+/H2VO4-/H2O2/picolinate (Pi-) system has been performed at 25 degrees C in 0.150 M Na(Cl) medium using quantitative 51 V NMR (500 MHz) and potentiometric data (glass electrode). In the ternary H+/H2VO4-/Pi- system, six complexes have been found in the pH region 1-10. In the quaternary H+/H2VO4-/H2O2/Pi- system, eight additional complexes have been found. Generally, equilibria are fast in both systems. The rate of peroxide decomposition depends on the species in solution. Chemical shifts, compositions and formation constants for the species are given. Equilibrium conditions and the fit of the model to the experimental data are illustrated in distribution diagrams. Possible formation of mixed ligand species with imidazole, lactic acid and citric acid have been investigated and ruled out under the same experimental conditions. Structural proposals are given, based on 1)C NMR data and available crystal structures.     

Identification of low molecular vaporizable components in silicone resins using gas chromatography/mass spectrometry

An experimental set-up for the detection of elemental chlorine in chlorinated hydrocarbons (CHCs) is described based on a miniaturized system, which could be used for on-line monitoring of chlorinated compounds. With an optimized time-resolved detection chlorine from CHCs like CCl₄ can be determined by Laser-Induced-Breakdown-Spectroscopy (LIBS) with g/g-detection limits in the gas phase. The application of a miniaturized Nd : YAG laser resulted only in a minor loss in performance, hence it could be used for designing a rugged and small on-line sensor. In addition, preliminary results for the detection of chlorine via the formation of CuCl in the plasma formed by focussing the laser on a copper surface are reported. Utilizing the luminescence of the CuCl D-system at 440 nm, a tenfold improvement in the detection limits was obtained. It appears that the formation of ad hoc selected, small molecules in a laser plasma could be a promising alternative for the selective and sensitive analysis of gaseous chlorinated and other species.     

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