Continuous-flow protease assay based on fluorescence resonance energy transfer

A homogeneous continuous-flow assay using fluorescence resonance energy transfer (FRET) for detection was developed to measure the hydrolysis of HIV Protease Substrate 1 (to which two choromophores, EDANS and DABCYL are covalently attached) by a protease (e.g. Subtilisin Carlsberg) and the influence of inhibitors. In the continuous-flow assay, an inhibitor solution and an enzyme solution were first eluted into the system and allowed to react with each other in a reaction coil. Subsequently, the substrate solution was added to an enzyme-inhibitor mixture in a second reaction coil and incubated for 1 min. Finally, the fluorescence intensity was monitored.The system was also utilized to measure the inhibition of the protease by two weak acidity inhibitors which are 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and ethylenediaminetetraacetic acid (EDTA). Using the obtained optimum conditions for AEBSF, a detection limit of 0.3 mmol/l was achieved and the relative standard deviation was below 3.7% in the 2.5-7.5 mmol/l range. For EDTA, which required a 20 times higher substrate concentration than AEBSF, a detection limit of 0.2 mmol/l was obtained and the relative standard deviation was below 9.6% in the 0.5-7.5 mmol/l range.The optimization of pH, substrate concentration, enzyme concentration, reaction time and temperature are described. Organic modifier effects were also investigated. Methanol, acetonitrile and DMSO could be tolerated up to 30%.     

Highly Active and Stable Pt-USY in the Low-Temperature de-NOx HC-SCR

Selective catalytic reduction of NO_x by propene has been investigated on Pt-USY and compared to other Pt-catalysts. The catalyst was characterized by XRD, Ar adsorption at 87 K, TEM, and CO chemisorption, and tested in a gas mixture system in excess oxygen. Pt-USY shows an excellent activity in the reaction, with a molar NO_x conversion of 90% at 475 K. Stability during time-on-stream and resistance to SO₂ and H₂O in the feed stream has also been investigated. Pt-USY performs better under lean-burn conditions than other Pt-catalysts on ZSM-5, Al₂O₃, or SiO₂. The selectivity to N₂ was similar for all the catalysts (30%), the other major product being N₂O.     

A correlation between 13C and 59Co NMR data and the catalytic activity of organocobalt complexes in the synthesis of pyridine derivatives

The correlation between the ¹³C and ⁵⁹Co NMR spectra of substituted cyclopentadienylcobalt complexes and their catalytic properties in the synthesis of pyridine derivatives is examined. Since the correlations can be expressed as linear relationships, a direct screening of potential catalysts by NMR is possible.     

Experiments at the Giessen source TEPOS with low energy positrons

The TEPOS facility at the Giessen LINAC delivers intense positron beams in the energy range between some eV and 6 keV; with postacceleration up to 80 keV. Results for remoderation and positron storage will be discussed. Further the energy-loss of positrons in thin aluminium foils at incident energies of 6–20 keV was measured. Cross sections for K- and L-shell ionization of thin silver and gold targets by positron and electron impact were determined at projectile energies of 30–70 keV. The experimental results are presented in detail; they are confirmed by calculations in plane wave Born approximation (PWBA) which include an electron exchange term and take into account the deceleration or acceleration of the incident projectile in the nuclear field of the target atom.     

Enantioselective room temperature phosphorescence detection of non-phosphorescent analytes based on interaction with beta-cyclodextrin/1-bromonaphthalene complexes

Menthol (MT) induces strong room temperature phosphorescence (RTP) of 1-bromonaphthalene (1BrN) in aqueous β-cyclodextrin (β-CD) suspensions, even under non-deoxygenated conditions. Interestingly, (−)-MT and (+)-MT enantiomers give rise to different phosphorescence intensities, the difference being 19 ± 3%. It is argued that the signal can be mainly ascribed to the formation of ternary complexes β-CD/1BrN/MT which show different RTP lifetimes, i.e. 4.28 ± 0.06 and 3.71 ± 0.06 ms for (−)-MT and (+)-MT, respectively. Most probably, the stereochemical structure of (−)-MT provides a better protection of 1BrN against quenching by oxygen than (+)-MT. This interpretation is in line with the observation that under deoxygenated conditions the phosphorescence intensity difference for the two complexes becomes very small, i.e. only about 4%.The lifetime difference under aerated conditions enables the direct determination of the MT stereochemistry. For mixtures, in view of the 0.06 ms uncertainty in the lifetime, enantiomeric purity can be determined down to 10%. Furthermore, in the case of MT the concentration of the least abundant enantiomer should be at least 3 × 10⁻⁴ M, since otherwise complex dissociation would obscure the lifetime difference.     

Measurement of the η production in proton-proton collisions with the COSY time-of-flight spectrometer

The reaction pp → ppη was measured at excess energies of 15 and 41 MeV at an external target of the Jülich Cooler Synchrotron COSY with the time-of-flight spectrometer. About 25000 events were measured for the excess energy of 15 MeV and about 8000 for 41 MeV. Both protons of the process ppη were detected with an acceptance of nearly 100% and the η was reconstructed by the missing-mass technique. For both excess energies the angular distributions are found to be nearly isotropic. In the invariant-mass distributions strong deviations from the pure phase space distributions are seen. Received: 24 May 2002 / Accepted: 20 September 2002 / Published online: 17 January 2003 RID="a" ID="a"e-mail: e.roderburg@fz-juelich.de Communicated by M. Gar?on     

Structural analysis of a melaminium polyphosphate from X-ray powder diffraction and solid-state NMR data

The crystal structure of the environmentally friendly flame retardant melaminium polyphosphate (MPoly) (2,4,6-triamino-1,3,5-triazinium x PO(3))(n)was determined by a direct-space global optimization technique from X-ray powder diffraction data. Solid-state NMR was used to corroborate the proposed hydrogen-bonding model and to determine the average degree of polymerization (n > 100). An analysis of the crystal structure of MPoly reveals aspects of molecular geometry and packing that are characteristic for melamine-containing compounds and polyphosphate salts. A comparison of MPoly with the crystal structures of its precursors melaminium orthophosphate (MP) and melaminium dihydrogenpyrophosphate (MPy) provides insight in the mechanism of the endothermic dehydration processes that takes place in the reaction path MP --> MPy --> MPoly. Solid-state NMR characterization of various samples of the same batch showed inhomogeneities in the MPoly composition. Various quantities of orthophosphates were found, which cannot be assigned to be MP.