Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3482. Adviser: Neil L. Kelleher. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph. D.)--Indiana University, Dept. of Chemistry, 2005. / Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6578. Adviser: David E. Clemmer. Title from dissertation home page (viewed Oct. 11, 2006).

<p> A direct solid sampling device has been developed using a theta pinch configuration to generate a pulsed plasma at atmospheric pressure. Energy from a 20kV, 1.80&micro;F capacitive discharge system is to inductively couple with the sacrificial aluminum thin film and produce a cylindrical plasma. Electrical simulations of the main discharge circuit were analyzed to determine the necessary circuit components that would withstand the <i>worst case scenario.</i> The design uses 4&rdquo; by 0.75&rdquo; copper stock at varying lengths to make the transmission lines and must also accommodate a spark gap switch and Rowgowski coil into the design. The 5.5 turn prototype coil design is used in initial testing to examine behavior of the system when discharged.</p>

Protein interactions are crucial for the survival of living organisms. The possibility of mapping the contact surfaces between proteins and their interacting partners is fundamental to understanding the mechanisms involved in the process. For these reasons techniques able to provide structural information on a short time scale and employing small amounts of material are sought after. The work reported in this thesis explores the use of carbene-based labelling in combination with mass spectrometry for protein footprinting and its applications in the study of protein structure and interactions. Studies on the efficiency and selectivity of a novel water-soluble photo-activated probe revealed its superior properties in comparison with diazirine-based reagents previously described for the same application. Using this methodology, the contact surface of the complex between lysozyme and NAG5, a carbohydrate substrate, was accurately mapped. The same technique was successfully employed to shed light on the structural change occurring to USP5, a large multi-domain deubiquitinating enzyme, upon its binding to diubiquitin. The use of carbene footprinting in combination with other biophysical techniques allowed to characterise the spatial arrangement of domains located at a module junction in the large multi-modular gladiolin polyketide synthase paving the way for future efforts by synthetic biologists to hijack the chemistry of this antibiotic-producing multiprotein enzyme to produce novel active compounds. The possibility of using carbene-based footprinting to gain insight into the structure of integral membrane proteins was also explored. The probe introduced in this work exhibited peculiar labelling properties when activated in the presence of a detergent-solubilised membrane protein. The reagent selectively reacted with portion of the protein in contact with detergent molecules showing potential to elucidate the quaternary structure of multimeric membrane proteins.

A very simple microscopic technique has been developed for the observation of uni-molecular steps. This technique utilises the diffraction effect at the edge of such steps, and steps of 4.6 A and 7.9 A have been observed on beryl crystals. This effect is discussed and a theoretical account given of methods of microscopic examination likely to result in the greatest sensitivity. It is concluded that steps of 1 A , or less, should be visible when reflexion phase contrast equipment is used. A review is given of past work on crystal growth in which it is shown that only the dislocation theory of growth is capable of satisfactorily explaining much of the experimental data. The microscopic diffraction technique and multiple beam interferometric techniques are applied to the study of the topography of faces of beryl and quartz crystals. As the microscopic technique enables one to observe dislocations directly, experimental proof is given that these crystals have grown by a dislocation mechanism, and that the dislocation theory of growth must therefore be generally applicable. This theory is shown to be correct in its many detailed predictions and quantitative information is obtained about the conditions in which some of these crystals have grown. The activity of dislocation groups, the cross-linking of layers, the "bunching" of layers, and "rod-formation" on beryl, are discussed. The treatment by the dislocation theory of dissolution as the simple reverse of the growth process is shown to be correct. Information is obtained on the density of dislocations and they are shown to occur frequently on transition surfaces and limited slip-zones. Direct visual proof is given that the dislocation theory of slip is correct. The topography of a cleavage face of topaz is briefly discussed.

Methods for both the on-line and off-line fluorimetric determination of total selenium and selenium species using 2-(alpha-pyridyl)-thioquinaldinamide (PTQA) as a fluorimetric reagent have been developed. As PTQA is not available on the market, methods for its synthesis were critically reviewed, and in the light of the information obtained, modifications were made to the synthesis and purification conditions in order to improve yield from 16 % to 38.9 %. The synthesised PTQA was used for the optimisation of a batch fluorimetric method based on the reaction between the reagent and Se (IV) in acidic medium. The developed method was applied to the determination of total selenium in nutritional supplements after microwave digestion and photoreduction of Se (VI) to Se (IV). The analytical precision in the concentration range 0. 5 - 1000 mug ml[-1] was better than 5%. Excellent agreement (r[2] = 0.9960) between the results of the fluorimetric and ICP-MS methods was obtained when a reference material, TMDA 51.2 supplied by National Water Research Institute Environment Canada and three nutritional supplements were analysed. A novel automated fluorimetric method for the simultaneous determination of Se (VI) and Se (IV) is described in which a purpose built flow injection system was used for the on-line photoreduction of Se (VI) to Se (IV) before detection. Variables such as tube diameter, flow rates, type of acids (HNO[3], H[2]SO[4], CH[3]COOH, H[3]PO[4] and HCI) that affect the rate at which PTQA reacted with Se (IV) were investigated and optimised. The linear calibration range was found to depend on the type and concentration of acid. The most intense fluorescence signals were observed in HNO3 and at a PTQA concentration of 5x10[-4] M. A sample throughput of 8 samples/hr was achieved. Method validation was by comparing the results obtained for the determination of selenium in nutritional supplements with those from ICP-MS.Selenium speciation in the nutritional supplements was studied using a developed method in which the selenium species selenomethionine, Se (IV), and Se (VI) were extracted, separated by HPLC prior to on-line photoreduction of Se (VI), and post column hydride generation before ICP-MS detection. Separation of the three species was achieved with excellent resolution in less than 18 minutes. The limit of determination for the three species was 0.50 mug L[-1].

Flow Injection (FI) techniques for use with Atomic Absorption Spectrometry (AAS) have attracted considerable research interest for sample introduction and calibration strategies. The interest continues in on-line preconcentration and matrix modification which are carried out by the incorporation of liquid-liquid extraction, ion-exchange, immobilised reagents, gas/vapour generation and continuous precipitation-filtration devices into suitably designed manifolds. Indirect determination of inorganic anions and organic compounds is becoming routine by FI–flame AAS. The direct analysis of solid samples, which is a shortcoming of flame AAS, was recently carried out by on-line electrolytic dissolutions.

<p> Conformational heterogeneity and protein dynamics play important roles in molecular recognition, but are experimentally difficult to characterize with sufficient temporal and spatial resolution. Infrared (IR) spectroscopy can probe protein dynamics on sub-ps timescales; and further, the small size of vibrational chromophores combined with site-selective incorporation of spectrally isolated IR probes provides high spatial resolution. Herein, we site-specifically introduce nitrile and carbon-deuterium bonds at distinct sites in the Src-homology 3 (SH3) domain from yeast protein Sho1 and its proline-rich peptide binding partner from Pbs2 to examine the underlying mechanisms of molecular recognition via IR spectroscopy. Further, we present efforts at developing instrumentation aimed at improving characterization of weakly absorbing vibrational probes in strongly absorbing solvent.</p><p> Nitrile probes were introduced at six distinct sites in the SH3 domain via amber codon suppression. Variation between the observed absorbance bands indicates site specific differences in conformational heterogeneity imposed by protein domain. Residue-specific changes upon peptide binding are observed at incorporated nitrile moieties, but are more dramatically observed for deuterated vibrational probes incorporated within the peptide binding partner. Deuterated amino acids were incorporated at highly conserved proline residues within the peptide ligand. Upon binding, absorbance bands are observed which indicate population of multiple conformational states in the bound complex. Only single resonances were observed by characterization of the same labeled bonds by NMR, suggesting rapid interconversion on the NMR timescale. Results suggest that the SH3 domain recognizes its peptide binding partner with at least elements of an induced-fit mechanism.</p><p> Characterization of the vibrational probes used above can be challenging due to the path length limitation imposed by the presence of strongly absorbing solvent water. This places an upper bound on the achievable signal strength which can obscure small (&micro;OD) absorbance bands. To confront this limitation we have constructed an absorbance spectrometer with a quantum cascade laser (QCL) source. The instrument allows characterization of samples of increased path length with similar signal-to-noise ratios as in FT IR measurements. Achievable signal-to-noise ratios are limited by QCL source noise; we present several approaches, one electronic and one interferometric, aimed at limiting the deleterious effect of QCL fluctuations.</p><p>

Raman spectroscopy is potentially a useful technique for the analysis of thioethers and thiols because of the characteristic set of bands which appears in the C-S stretching region. A review of previous studies on Raman band intensity measurements is presented in which the experimental difficulties and influencing factors are given. An experimental system has been developed around a Coderg PHO Raman spectrometer for the measurement of absolute Raman band intensities. This includes the novel use of a Pockels cell for the accurate determination of depolarisation ratios. Carbon tetrachloride has been assessed as a suitable internal standard for use with this system. Raman band intensities are given for five thioethers and two thiols. Several bands are identified as being characteristic of sulphur-containing hydrocarbons. Analysis of bands in the C-S stretching region is accomplished by considering the intensity contributions provided by individual groups in the molecule, resulting in a successful prediction of the C-S stretching band intensities for t-butane thiol. In addition, temperature studies have been conducted for ethyl methyl sulphide in order to attempt calculation of the energy barrier between the two conformations. A computer programme has been developed for the theoretical calculation of Raman band frequencies and intensities. Molecular parameters have been derived for dimethyl sulphide, including an ab initio force field. Tests for transferabilityof these data to ethyl methyl sulphide and deuterated dimethyl sulphide have produced encouraging results. A study of infra-red intensities of thioethers has been carried out. This is an extension of some previous work in which the necessity to resolve individual bands is eliminated. For several discrete spectral regions quantification is made of the effects of a sulphur atom on C-H band intensities, thus allowing successful intensity predictions for other molecules studied. Suggestions for further work in this field are given.

The use of immobilized enzymes (and cells) in flow analyses and some determinants of the performance of the resulting analytical systems have been examined; the detector used in most cases was the LKB 2017-30 flow microcalorimeter. The detector time constant and sensitivity, reactor enzyme activity, flow rate and the mode of sample injection were primary determinants of system characteristics such as, sensitivity, minimum detectable concentration, linearity range and speed analysis. These relationships were later applied in the determination of the reaction enthalpy. Glucose, urea, fructose, mannose, glucoseamine, the cofactor ATP, vitamin C and hydrogen peroxide were also determined using the appropriate immobilized enzymes. The kinetics of the reactions catalysed by immobilized urease, glucose oxidase, acetylcholinesterase or ascorbate oxidase was studied via flow microcalorimetry. Apparent intrinsic kinetic parameters, external and internal substrate moduli, as well as the corresponding effectiveness factors were determined. In all cases,the Michaelis constant (Km) was increased and the observed activity severely limited as a consequence of the slow diffusion of substrate. Flow analysis using immobilized cells and microcalorimetric monitoring was also examined. This combination is well suited for broad-spectrum analysis. The effects of selected organic solvents (permeablizing agents) on the linearity range (approximately given by the Km) was also of some interest. The specificity (estimated as (dQ/dt)max/KmaPP) for a range of substrate was proposed as a means of differentiating between closely related strains of cells. Electrochemical monitoring of immobilized enzyme reactions may rival the thermochemical approach in its widespread applicability and simplicity. Both potentiometric and amperometric modes were used; the latter in connection with flow analyses. Two amperometric enzyme reactors and a flow-through enzyme electrode were constructed and successfully used in substrate determinations.