State defining experiment in chemical kinetics. Primary characterization of catalyst activity in a TAP experiment

This paper presents a new strategy, “state-by-state transient screening”, for kinetic characterization of states of a multicomponent catalyst as applied to TAP pulse-response experiments. The key idea is to perform an insignificant chemical perturbation of the catalytic system so that the known essential characteristics of the catalyst (e.g. oxidation degree) do not change during the experiment. Two types of catalytic substances can be distinguished: catalyst state substances, which determine the catalyst state, and catalyst dynamic substances, which are created by the perturbation. The general methodological and theoretical framework for multi-pulse TAP experiments is developed, and the general model for a one-pulse TAP experiment is solved. The primary kinetic characteristics, basic kinetic coefficients, are extracted from diffusion–reaction data and calculated as functions of experimentally measured exit-flow moments without assumptions regarding the detailed kinetic mechanism. The new strategy presented in this paper provides essential information, which can be a basis for developing a detailed reaction mechanism. The theoretical results are illustrated using furan oxidation over a VPO catalyst.

Kinetics of phase transformations in a model with metastable fluid-fluid separation: A molecular dynamics study

By molecular dynamics (MD) simulations we study the crystallization process in a model system whose particles interact by a spherical pair potential with a narrow and deep attractive well adjacent to a hard repulsive core. The phase diagram of the model displays a solid-fluid equilibrium, with a metastable fluid-fluid separation. Our computations are restricted to fairly small systems (from 2592 to 10368 particles) and cover long simulation times, with constant energy trajectories extending up to 76x10(6) MD steps. By progressively reducing the system temperature below the solid-fluid line, we first observe the metastable fluid-fluid separation, occurring readily and almost reversibly upon crossing the corresponding line in the phase diagram. The nucleation of the crystal phase takes place when the system is in the two-fluid metastable region. Analysis of the temperature dependence of the nucleation time allows us to estimate directly the nucleation free energy barrier. The results are compared with the predictions of classical nucleation theory. The critical nucleus is identified, and its structure is found to be predominantly fcc. Following nucleation, the solid phase grows steadily across the system, incorporating a large number of localized and extended defects. We discuss the relaxation processes taking place both during and after the crystallization stage. The relevance of our simulation for the kinetics of protein crystallization under normal experimental conditions is discussed. (C) 2002 American Institute of Physics.

The production and characterisation of an antibody to detect the coccidiostat toltrazuril and its metabolite ponazuril.

The production of an antibody to detect toltrazuril or its metabolite ponazuril is complicated due to structural constraints of conjugating these coccidiostats to a carrier protein. Therefore a search was carried out for a compound that shared a common substructure to use as an antigen mimic. The chosen compound, trifluoraminoether, was conjugated to two carrier proteins (HSA and BTG) and used in the immunisation of six rabbits. Two immunogen doses (1 mg and 0.1 mg) were also used. All six rabbits produced an immunological response to the hapten regardless of the carrier protein or immunogen dose used. The most sensitive polyclonal antibody produced, designated R609, was subsequently characterised. This antiserum exhibited an IC50 of 18 ng ml-1 using a competitive ELISA format. Cross reactivity studies show that this serum is specific for toltrazuril and its metabolites (toltrazuril sulfoxide and toltrazuril sulfone) but does not cross-react with other coccidiostats such as halofuginone, nitroimidazoles or nicarbazin. This is the first reported production of an antibody capable of specifically binding toltrazuril and ponazuril.

Structure and kinetics of phosphonopyruvate hydrolase from Variovorax sp. Pal2: New insight into the divergence of catalysis within the PEP mutase/isocitrate lyase superfamily .

Phosphonopyruvate (P-pyr) hydrolase (PPH), a member of the phosphoenolpyruvate (PEP) mutase/isocitrate lyase (PEPM/ICL) superfamily, hydrolyzes P-pyr and shares the highest sequence identity and functional similarity with PEPM. Recombinant PPH from Variovorax sp. Pal2 was expressed in Escherichia coli and purified to homogeneity. Analytical gel filtration indicated that the protein exists in solution predominantly as a tetramer. The PPH pH rate profile indicates maximal activity over a broad pH range.The steady-state kinetic constants determined for a rapid equilibrium ordered kinetic mechanism with Mg+2 binding first (Kd =140 ± 40 M), are kcat = 105 ± 2 s-1 and P-pyr Km = 5 ± 1 M. PEP (slow substrate kcat = 2 × 10-4 s-1), oxalate, and sulfopyruvate are competitive inhibitors with Ki values of 2.0 ± 0.1 mM, 17 ± 1 M, and 210 ± 10 M, respectively. Three PPH crystal structures have been determined, that of a ligand-free enzyme, the enzyme bound to Mg2+ and oxalate (inhibitor), and the enzyme bound to Mg2+ and P-pyr (substrate). The complex with the inhibitor was obtained by cocrystallization, whereas that with the substrate was obtained by briefly soaking crystals of the ligand-free enzyme with P-pyr prior to flash cooling. The PPH structure resembles that of the other members of the PEPM/ICL superfamily and is most similar to the functionally related enzyme, PEPM. Each monomer of the dimer of dimers exhibits an (/)8 barrel fold with the eighth helix swapped between two molecules of the dimer. Both P-pyr and oxalate are anchored to the active site by Mg2+. The loop capping the active site is disordered in all three structures, in contrast to PEPM, where the equivalent loop adopts an open or disordered conformation in the unbound state but sequesters the inhibitor from solvent in the bound state. Crystal packing may have favored the open conformation of PPH even when the enzyme was cocrystallized with the oxalate inhibitor. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop. The proposed PPH catalytic mechanism is analogous to that of PEPM but includes activation of a water nucleophile with the loop Thr118 residue.

Evaluation of predicted network modules in yeast metabolism using NMR-based metabolite profiling

Genome-scale metabolic models promise important insights into cell function. However, the definition of pathways and functional network modules within these models, and in the biochemical literature in general, is often based on intuitive reasoning. Although mathematical methods have been proposed to identify modules, which are defined as groups of reactions with correlated fluxes, there is a need for experimental verification. We show here that multivariate statistical analysis of the NMR-derived intra- and extracellular metabolite profiles of single-gene deletion mutants in specific metabolic pathways in the yeast Saccharomyces cerevisiae identified outliers whose profiles were markedly different from those of the other mutants in their respective pathways. Application of flux coupling analysis to a metabolic model of this yeast showed that the deleted gene in an outlying mutant encoded an enzyme that was not part of the same functional network module as the other enzymes in the pathway. We suggest that metabolomic methods such as this, which do not require any knowledge of how a gene deletion might perturb the metabolic network, provide an empirical method for validating and ultimately refining the predicted network structure.

Studies of switching kinetics in ferroelectric thin films

Experimental studies are reported concerning the importance of interfacial capacitance (including electrode screening, space-charge layers, and/or chemically discrete dead layers). on domain switching behaviour in thin films of ferroelectric lead zirconate-titanate (PZT), strontium bismuth tantalate (SBT), and barium strontium titanate (BST). Emphasis is placed upon studies at applied field values very near the coercive field E, asymmetry in fatigue for positive and negative polarity coercive fields, and in the case of BST, of the coexistence of ferroelectric and paraelectric phases Studies of dielectric loss show important correlations between tan 6 and fatigue (polarization decrease) as a function of bipolar switching cycles N. This is a priori not obvious, since the former is a linear response and the latter, a nonlinear response. Modelling of enlarged interfacial,space-charge layers in PZT films and chemically distinct dead (paraelectric) layers in BST films shows contradictory tendencies of coercive-voltage changes with the growth of passive layers.