Reactivity characteristics of cytochrome c, cytochrome oxidase and the electrostatic cytochrome c - cytochrome oxidase complex /

The mechanism whereby cytochrome £ oxidase catalyses elec-. tron transfer from cytochrome £ to oxygen remains an unsolved problem. Polarographic and spectrophotometric activity measurements of purified, particulate and soluble forms of beef heart mitochondrial cytochrome c oxidase presented in this thesis confirm the following characteristics of the steady-state kinetics with respect to cytochrome £: (1) oxidation of ferrocytochrome c is first order under all conditions. -(2) The relationship between sustrate concentration and velocity is of the Michaelis-Menten type over a limited range of substrate. concentrations at high ionic strength. (3) ~he reaction rate is independent from oxygen concentration until very low levels of oxygen. (4) "Biphasic" kinetic plots of enzyme activity as a function of substrate concentration are found when the range of cytochrome c concentrations is extended; the biphasicity ~ is more apparent in low ionic strength buffer. These results imply two binding sites for cytochrome £ on the oxidase; one of high affinity and one of low affinity with Km values of 1.0 pM and 3.0 pM, respectively, under low ionic strength conditions. (5) Inhibition of the enzymic rate by azide is non-c~mpetitive with respect to cytochrome £ under all conditions indicating an internal electron transfer step, and not binding or dissociation of £ from the enzyme is rate limiting. The "tight" binding of cytochrome '£ to cytochrome c oxidase is confirmed in column chromatographic experiments. The complex has a cytochrome £:oxidase ratio of 1.0 and is dissociated in media of high ionic strength. Stopped-flow spectrophotometric studies of the reduction of equimolar mixtures and complexes of cytochrome c and the oxidase were initiated in an attempt to assess the functional relevance of such a complex. Two alternative routes -for reduction of the oxidase, under conditions where the predominant species is the £ - aa3 complex, are postulated; (i) electron transfer via tightly bound cytochrome £, (ii) electron transfer via a small population of free cytochrome c interacting at the "loose" binding site implied from kinetic studies. It is impossible to conclude, based on the results obtained, which path is responsible for the reduction of cytochrome a. The rate of reduction by various reductants of free cytochrome £ in high and low ionic strength and of cytochrome £ electrostatically bound to cytochrome oxidase was investigated. Ascorbate, a negatively charged reagent, reduces free cytochrome £ with a rate constant dependent on ionic strength, whereas neutral reagents TMPD and DAD were relatively unaffected by ionic strength in their reduction of cytochrome c. The zwitterion cysteine behaved similarly to uncharged reductants DAD and TI~PD in exhibiting only a marginal response to ionic strength. Ascorbate reduces bound cytochrome £ only slowly, but DAD and TMPD reduce bound cytochrome £ rapidly. Reduction of cytochrome £ by DAD and TMPD in the £ - aa3 complex was enhanced lO-fold over DAD reduction of free £ and 4-fold over TMPD reduction of free c. Thus, the importance of ionic strength on the reactivity of cytochrome £ was observed with the general conclusion being that on the cytochrome £ molecule areas for anion (ie. phosphate) binding, ascorbate reduction and complexation to the oxidase overlap. The increased reducibility for bound cytochrome £ by reductants DAD and TMPD supports a suggested conformational change of electrostatically bound c compare.d to free .£. In addition, analysis of electron distribution between cytochromes £ and a in the complex suggest that the midpotential of cytochrome ~ changes with the redox state of the oxidase. Such evidence supports models of the oxidase which suggest interactions within the enzyme (or c - enzyme complex) result in altered midpoint potentials of the redox centers.

MS Measurements

RMS measuring device is a nonlinear device consisting of linear and nonlinear devices. The performance of rms measurement is influenced by a number of factors; i) signal characteristics, 2) the measurement technique used and 3) the device characteristics. RMS measurement is not simple, particularly when the signals are complex and unknown. The problem of rms measurement on high crest-factor signals is fully discussed and a solution to this problem is presented in this thesis. The problem of rms measurement is systematically analized and found to have mainly three types of errors: (1) amplitude or waveform error 2) Frequency error and (3) averaging error. Various rms measurement techniques are studied and compared. On the basis of this study the rms -measurement is reclassified three categories: (1) Wave-form-error-free measurement (2) High-frequncy-error measurement and (3) Low-frequency error-free measurement. In modern digital sampled-data systems the signals are complex and waveform-error-free rms measurement is highly appreciated. Among the three basic blocks of rms measuring device the squarer is the most important one. A squaring technique is selected, that permits shaping of the squarer error characteristic in such a way as to achieve waveform-errob free rms measurement. The squarer is designed, fabricated and tested. A hybrid rms measurement using an analog rms computing device and digital display combines the speed of analog techniques and the resolution and ease of measurement of digital techniques. An A/D converter is modified to perform the square-rooting operation. A 10-V rms voltmeter using the developed rms detector is fabricated and tested. The chapters two, three and four analyse the problems involved in rms measurement and present a comparative study of rms computing techniques and devices. The fifth chapter gives the details of the developed rms detector that permits wave-form-error free rms measurement. The sixth chapter, enumerates the the highlights of the thesis and suggests a list of future projects

Optical Characterization of Organic Gain Materials for UV-emitting Hybrid Complex Coupled VCSELs

In this thesis, optical gain measurement setup based on variable stripe length method is designed, implemented and improved. The setup is characterized using inorganic and organic samples. The optical gain of spiro-quaterphenyl is calculated and compared with measurements from the setup. Films with various thicknesses of spiro-quaterphenyl, methoxy-spiro-quaterphenyl and phenoxy-spiro-quaterphenyl are deposited by a vacuum vapor deposition technique forming asymmetric slab waveguides. The optical properties, laser emission threshold, optical gain and loss coefficient for these films are measured. Additionally, the photodegradation during pumping process is investigated.

Estimation of zero-sequence impedance of undergrounds cables for single-phase fault location in distribution systems with electric arc

This paper focus on the problem of locating single-phase faults in mixed distribution electric systems, with overhead lines and underground cables, using voltage and current measurements at the sending-end and sequence model of the network. Since calculating series impedance for underground cables is not as simple as in the case of overhead lines, the paper proposes a methodology to obtain an estimation of zero-sequence impedance of underground cables starting from previous single-faults occurred in the system, in which an electric arc occurred at the fault location. For this reason, the signal is previously pretreated to eliminate its peaks voltage and the analysis can be done working with a signal as close as a sinus wave as possible

Localización de faltas en sistemas de distribución de energía eléctrica usando métodos basados en el modelo y métodos basados en el conocimiento

La calidad de energía eléctrica incluye la calidad del suministro y la calidad de la atención al cliente. La calidad del suministro a su vez se considera que la conforman dos partes, la forma de onda y la continuidad. En esta tesis se aborda la continuidad del suministro a través de la localización de faltas. Este problema se encuentra relativamente resuelto en los sistemas de transmisión, donde por las características homogéneas de la línea, la medición en ambos terminales y la disponibilidad de diversos equipos, se puede localizar el sitio de falta con una precisión relativamente alta. En sistemas de distribución, sin embargo, la localización de faltas es un problema complejo y aún no resuelto. La complejidad es debida principalmente a la presencia de conductores no homogéneos, cargas intermedias, derivaciones laterales y desbalances en el sistema y la carga. Además, normalmente, en estos sistemas sólo se cuenta con medidas en la subestación, y un modelo simplificado del circuito. Los principales esfuerzos en la localización han estado orientados al desarrollo de métodos que utilicen el fundamental de la tensión y de la corriente en la subestación, para estimar la reactancia hasta la falta. Como la obtención de la reactancia permite cuantificar la distancia al sitio de falta a partir del uso del modelo, el Método se considera Basado en el Modelo (MBM). Sin embargo, algunas de sus desventajas están asociadas a la necesidad de un buen modelo del sistema y a la posibilidad de localizar varios sitios donde puede haber ocurrido la falta, esto es, se puede presentar múltiple estimación del sitio de falta. Como aporte, en esta tesis se presenta un análisis y prueba comparativa entre varios de los MBM frecuentemente referenciados. Adicionalmente se complementa la solución con métodos que utilizan otro tipo de información, como la obtenida de las bases históricas de faltas con registros de tensión y corriente medidos en la subestación (no se limita solamente al fundamental). Como herramienta de extracción de información de estos registros, se utilizan y prueban dos técnicas de clasificación (LAMDA y SVM). Éstas relacionan las características obtenidas de la señal, con la zona bajo falta y se denominan en este documento como Métodos de Clasificación Basados en el Conocimiento (MCBC). La información que usan los MCBC se obtiene de los registros de tensión y de corriente medidos en la subestación de distribución, antes, durante y después de la falta. Los registros se procesan para obtener los siguientes descriptores: a) la magnitud de la variación de tensión ( dV ), b) la variación de la magnitud de corriente ( dI ), c) la variación de la potencia ( dS ), d) la reactancia de falta ( Xf ), e) la frecuencia del transitorio ( f ), y f) el valor propio máximo de la matriz de correlación de corrientes (Sv), cada uno de los cuales ha sido seleccionado por facilitar la localización de la falta. A partir de estos descriptores, se proponen diferentes conjuntos de entrenamiento y validación de los MCBC, y mediante una metodología que muestra la posibilidad de hallar relaciones entre estos conjuntos y las zonas en las cuales se presenta la falta, se seleccionan los de mejor comportamiento. Los resultados de aplicación, demuestran que con la combinación de los MCBC con los MBM, se puede reducir el problema de la múltiple estimación del sitio de falta. El MCBC determina la zona de falta, mientras que el MBM encuentra la distancia desde el punto de medida hasta la falta, la integración en un esquema híbrido toma las mejores características de cada método. En este documento, lo que se conoce como híbrido es la combinación de los MBM y los MCBC, de una forma complementaria. Finalmente y para comprobar los aportes de esta tesis, se propone y prueba un esquema de integración híbrida para localización de faltas en dos sistemas de distribución diferentes. Tanto los métodos que usan los parámetros del sistema y se fundamentan en la estimación de la impedancia (MBM), como aquellos que usan como información los descriptores y se fundamentan en técnicas de clasificación (MCBC), muestran su validez para resolver el problema de localización de faltas. Ambas metodologías propuestas tienen ventajas y desventajas, pero según la teoría de integración de métodos presentada, se alcanza una alta complementariedad, que permite la formulación de híbridos que mejoran los resultados, reduciendo o evitando el problema de la múltiple estimación de la falta.

First CRDS-measurements of water vapour continuum in the 940nm absorption band

Measurements of near-infrared water vapour continuum using continuous wave cavity ring down spectroscopy (cw- CRDS) have been performed at around 10611.6 and 10685:2 cm1. The continuum absorption coefficients for N2- broadening have been determined for two temperatures and wavenumbers. These results represent the first near-IR continuum laboratory data determined within the complex spectral environment in the 940nm water vapour band and are in reasonable agreement with simulations using the semiempirical CKD formulation.

A field study of factors influencing the concentrations of a traffic-related pollutant in the vicinity of a complex urban junction

The paper describes a field study focused on the dispersion of a traffic-related pollutant within an area close to a busy intersection between two street canyons in Central London. Simultaneous measurements of airflow, traffic flow and carbon monoxide concentrations ([CO]) are used to explore the causes of spatial variability in [CO] over a full range of background wind directions. Depending on the roof-top wind direction, evidence of both flow channelling and recirculation regimes were identified from data collected within the main canyon and the intersection. However, at the intersection, the merging of channelled flows from the canyons increased the flow complexity and turbulence intensity. These features, coupled with the close proximity of nearby queuing traffic in several directions, led to the highest overall time-average measured [CO] occurring at the intersection. Within the main street canyon, the data supported the presence of a helical flow regime for oblique roof-top flows, leading to increased [CO] on the canyon leeward side. Predominant wind directions led to some locations having significantly higher diurnal average [CO] due to being mostly on the canyon leeward side during the study period. For all locations, small changes in the background wind direction could cause large changes in the in-street mean wind angle and local turbulence intensity, implying that dispersion mechanisms would be highly sensitive to small changes in above roof flows. During peak traffic flow periods, concentrations within parallel side streets were approximately four times lower than within the main canyon and intersection which has implications for controlling personal exposure. Overall, the results illustrate that pollutant concentrations can be highly spatially variable over even short distances within complex urban geometries, and that synoptic wind patterns, traffic queue location and building topologies all play a role in determining where pollutant hot spots occur.

Thermopile radiometer signal conditioning for surface atmospheric radiation measurements

A highly stable microvolt amplifier for use with atmospheric broadband thermopile radiometers is described. The amplifier has a nominal gain of 500, for bipolar input signals in the range +/- 10 mV from a floating source. The noise level at the input is less than 5 mu V (at 100 k Omega input impedance), permitting instantaneous diffuse solar radiation measurements to 0.5 W m(-2) resolution with 12 bit analog to digital conversion. The temperature stability of gain is better than 5 ppm/degrees C (-4 to 20 degrees C). Averaged over a decade of use, the long term drift of the amplifier gain is less than similar to 0.02%/yr. As well as radiometers measuring solar and terrestrial radiations, the amplifier has also been successfully used with low level signals from thermocouples and ground heat flux plates.

Doppler lidar measurements of oriented planar ice crystals falling from supercooled and glaciated layer clouds

The properties of planar ice crystals settling horizontally have been investigated using a vertically pointing Doppler lidar. Strong specular reflections were observed from their oriented basal facets, identified by comparison with a second lidar pointing 4° from zenith. Analysis of 17 months of continuous high-resolution observations reveals that these pristine crystals are frequently observed in ice falling from mid-level mixed-phase layer clouds (85% of the time for layers at −15 °C). Detailed analysis of a case study indicates that the crystals are nucleated and grow rapidly within the supercooled layer, then fall out, forming well-defined layers of specular reflection. From the lidar alone the fraction of oriented crystals cannot be quantified, but polarimetric radar measurements confirmed that a substantial fraction of the crystal population was well oriented. As the crystals fall into subsaturated air, specular reflection is observed to switch off as the crystal faces become rounded and lose their faceted structure. Specular reflection in ice falling from supercooled layers colder than −22 °C was also observed, but this was much less pronounced than at warmer temperatures: we suggest that in cold clouds it is the small droplets in the distribution that freeze into plates and produce specular reflection, whilst larger droplets freeze into complex polycrystals. The lidar Doppler measurements show that typical fall speeds for the oriented crystals are ≈ 0.3 m s−1, with a weak temperature correlation; the corresponding Reynolds number is Re ∼ 10, in agreement with light-pillar measurements. Coincident Doppler radar observations show no correlation between the specular enhancement and the eddy dissipation rate, indicating that turbulence does not control crystal orientation in these clouds. Copyright © 2010 Royal Meteorological Society

A novel Bayesian approach to drug design with simple and complex enzymes: Use with lens aldehyde dehydrogenase and the glyoxalase pathway

Purpose: Acquiring details of kinetic parameters of enzymes is crucial to biochemical understanding, drug development, and clinical diagnosis in ocular diseases. The correct design of an experiment is critical to collecting data suitable for analysis, modelling and deriving the correct information. As classical design methods are not targeted to the more complex kinetics being frequently studied, attention is needed to estimate parameters of such models with low variance. Methods: We have developed Bayesian utility functions to minimise kinetic parameter variance involving differentiation of model expressions and matrix inversion. These have been applied to the simple kinetics of the enzymes in the glyoxalase pathway (of importance in posttranslational modification of proteins in cataract), and the complex kinetics of lens aldehyde dehydrogenase (also of relevance to cataract). Results: Our successful application of Bayesian statistics has allowed us to identify a set of rules for designing optimum kinetic experiments iteratively. Most importantly, the distribution of points in the range is critical; it is not simply a matter of even or multiple increases. At least 60 % must be below the KM (or plural if more than one dissociation constant) and 40% above. This choice halves the variance found using a simple even spread across the range.With both the glyoxalase system and lens aldehyde dehydrogenase we have significantly improved the variance of kinetic parameter estimation while reducing the number and costs of experiments. Conclusions: We have developed an optimal and iterative method for selecting features of design such as substrate range, number of measurements and choice of intermediate points. Our novel approach minimises parameter error and costs, and maximises experimental efficiency. It is applicable to many areas of ocular drug design, including receptor-ligand binding and immunoglobulin binding, and should be an important tool in ocular drug discovery.

Equilibrium studies on mixed ligand complex formation of Co(II), Ni(II), Cu(II) and Zn(II) with N-(2-hydroxybenzyl)-L-histidine (H(2)hb-L-his) and typical N,N donor ligands: Crystal structure of [Ni(hb-L-his)(bipyridine)]center dot H2O complex

Equilibrium study on complex formation of Co(II), Ni(II), Cu(II) and Zn(II), hereafter M(II), with the quadridentate (O-, N, O-, N) donor ligand, N-(2-hydroxybenzyl)-L-histidine (H(2)hb-L-his, hereafter H2L), in the absence and in the presence of typical (N, N) donor bidentate ligands, 1,10 phenanthroline(phen), 2, 2'-bipyridine(bipy), ethylenediamine(en), hereafter B, in aqueous solution at 25 +/- 1 degrees C was done at a fixed ionic strength, I = 0.1 mol dm(-3) (NaNO3) by combined pH-metric, UV-Vis and EPR measurements provide evidence for the formation of mononuclear and dinuclear binary and mixed ligand complexes of the types: M(L), M(L)(2)(2-), M-2(L)(2+), M-2(H-1L)(+), M(L)(B), (B)M(H-1L)M(B)(+). The imidazole moiety of the ligand is found to act as a bridging bidentate ligand in the dinuclear M-2(L)(2+), M-2(H-1L)(+) and (B)M(H-1L)M(B)(+) complexes, using its N-3 atom and N1-H deprotonated moiety. Stability constants of the complexes provide evidence of discrimination of Cu(II) from the other M(II) ions by this ligand. Solid complexes: [Ni(L)(H2O)(2)] (1), [Cu(L)(H2O)] (2), and [Ni(L)(bipy)] (.) H2O (3) have been isolated and characterized by various physicochemical studies. Single crystal X-ray diffraction of the ternary complex, 3, shows an octahedral [(O-,N,N,O-)(N,N)] geometry with extensive pi-pi stacking of the aromatic rings and H-bonding with imidazole (N1-H), secondary amino N-atom, the lattice H2O molecule, and the carboxylate and phenolate O-atoms. (c) 2006 Elsevier B.V. All rights reserved.

Concomitant polymorphism of an antiferromagnetically coupled dicopper(II,II) complex with single strand helical assembly: synthesis, structure, DSC, magnetic and heterogeneous catalytic studies

Two concomitant polymorphic coordination complexes (dark blue - I and black - II) with the formula (Cu2C44H60N4O4) have been synthesized and characterized crystallographically. Magnetic measurements show the presence of a strong antiferromagnetic interaction and the 2J value corresponds extremely well to the theoretically calculated one, indicating the fact that it follows nicely the magneto-structural relationship. Immobilization of the copper(II) complex I on a 2D-hexagonal mesoporous silica showed good catalytic efficiency in the liquid phase partial oxidation of olefins in the presence of TBHP as an oxidant. (C) 2009 Elsevier Ltd. All rights reserved.

Synthesis, structure and properties of cobalt(II/III) seleno-bisphenolate complexes containing NN bidentate co-ligands: kinetic studies on the reduction of a cobalt(III) complex by ascorbic acid in 80% MeCN-20% water

Two cobalt complexes, [Co(L-Se)(phen)]center dot CH2Cl2 (1) and [Co(L-Se)(N,N-Me(2)en)(CH3COO-)] (2) have been synthesized and characterized by elemental analyses, magnetic measurements, i.r. studies etc. Single crystal X- ray studies reveal that in complex (1) cobalt atom is in +2 oxidation state with trigonal bipyramidal geometry, while in complex (2) it is in +3 oxidation state and surrounded octahedrally. The asymmetric unit of complex (2) contains two crystallographically independent discrete molecules. Complex (1) was found to be paramagnetic with mu(eff) = 2.19 BM indicating a low spin cobalt(II) d(7) system, whereas complex (2) is found to be diamagnetic with cobalt(III) in low spin d(6) state. The kinetic studies on the reduction of (2) by ascorbic acid in 80% MeCN-20% H2O (v/v) at 25 degrees C reveal that the reaction proceeds through the rapid formation of inner-sphere adduct, probably by replacing the loosely coordinated AcO- group, followed by electron transfer in a slow step and is supported by a large Q (formation constant) value.

Gas-phase kinetics of chlorosilylene reactions. I. ClSiH + Me3SiH: absolute rate measurements and theoretical calculations for prototype Si−H insertion reactions

Time-resolved studies of chlorosilylene, CISiH, generated by the 193 nm laser flash photolysis of 1-chloro-1-silacyclopent-3-ene, have been carried out to obtain rate constants for its bimolecular reaction with trimethylsilane, Me3SiH, in the gas phase. The reaction was studied at total pressures up to 100 torr (with and without added SF6) over the temperature range 297-407 K. The rate constants were found to be pressure independent and gave the following Arrhenius equation: log(k/cm(3) molecule(-1) s(-1)) = (-13.97 +/- 0.25) + (12.57 +/- 1.64) kJ mol(-1)/RT In 10. The Arrhenius parameters are consistent with a mechanism involving an intermediate complex, whose rearrangement is the rate-determining step. Quantum chemical calculations of the potential energy surface for this reaction and also the reactions of CISiH with SiH4 and the other methylsilanes support this conclusion. Comparisons of both experiment and theory with the analogous Si-H insertion processes of SiH2 and SiMe2 show that the main factor causing the lower reactivity of ClSiH is the secondary energy barrier. The calculations also show the existence of a novel intramolecular H-atom exchange process in the complex of ClSiH with MeSiH3.

Synthesis, X-ray crystal structures and properties of complex salts and sterically crowded heteroleptic complexes of group 10 metal ions with aromatic sulfonyl dithiocarbimates and triphenylphosphine ligand

Two new complex salts of the form (Bu4N)(2)[Ni(L)(2)] (1) and (Ph4P)(2)[Ni(L)(2)] (2) and four heteroleptic complexes cis-M(PPh3)(2)(L) [M = Ni(II) (3), Pd(II) (4), L = 4-CH3OC6H4SO2N=CS2] and cis-M(PPh3)(2)(L') [M = Pd(II) (5), Pt(II) (6), L' = C6H5SO2N=CS2] were prepared and characterized by elemental analyses, IR, H-1, C-13 and P-31 NMR and UV-Vis spectra, solution and solid phase conductivity measurements and X-ray crystallography. A minor product trans-Pd(PPh3)(2)(SH)(2), 4a was also obtained with the synthesis of 4. The NiS4 and MP2S2 core in the complex salts and heteroleptic complexes are in the distorted square-plane whereas in the trans complex, 4a the centrosymmetric PdS2P2 core is perforce square planar. X-ray crystallography revealed the proximity of the ortho phenyl proton of the PPh3 ligand to Pd(II) showing rare intramolecular C-H center dot center dot center dot Pd anagostic binding interactions in the palladium cis-5 and trans-4a complexes. The complex salts with sigma(rt) values similar to 10 (5) S cm (1) show semi-conductor behaviors. The palladium and platinum complexes show photoluminescence properties in solution at room temperature.