The theory of the quadrupole interaction in nuclear magnetic resonance spectroscopy and relaxation measurements is presented in detail, with applications to ²H-NMR studies of order and dynamics in bilayers of deuterated lipids. Investigations of lipid-protein interactions in reconstituted membrane systems and intact biological membranes are reviewed. An experimental program is described which uses a synthetic amphiphilic polypeptide, with known geometry and variable length, to isolate questions about the geometrical interpretation of orientational order in lipid-protein interactions. A report is presented of an investigation of the effects of this polypeptide, Lys₂-Gly-Leu₂₀-Lys₂-Ala-amide, on the mixed bilayer system: Dimyristoylphosphatidylcholine Di-per-deuterio-myristoylphosphatidic Acid. The addition of the peptide was found to have little effect (≤5%) on the first and second moments of the distribution of quadrupole splittings in the liquid crystalline phase. Similarly, the spin-lattice relaxation time constants were affected by ≤10% in the liquid crystalline phase. The time constant for the decay of the quadrupole echo decreased dramatically above the phase transition with the addition of peptide, a phenomenon which is explained in terms of the presence of a new slow motion in the lipid-peptide systems. A simple model of the slow motion induced by the peptide is proposed, in which the lipid molecules undergo a rapid exchange between boundary and bulk sites. An effective correlation time is determined from an average over the rotations on each of these sites. Using this model, estimates are made of the change in the second moment brought about by the onset of the rotations, and. of the number of binding sites on the peptide. These estimates are found to be in agreement with independent measurements of the change in the second moment, and the number of binding sites is within the range predicted by simple considerations of charge balance. The change in the lineshape with the variation of the spacing of the pulses in the quadrupole echo experiment was investigated, and it was determined that the transverse relaxation time constants have a slight orientation dependence. It was also determined that the addition of the peptide has no significant effect on the variation of the lineshape. Some experiments which could answer some of the questions raised by these results are suggested. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

The work described in this thesis deals mainly with aspects related to two- and three-dimensional NMR imaging. A detailed discussion on frequency-selective excitation using amplitude modulated rf pulses in relation to slice selection in NMR imaging has been presented. This includes the analysis and implementation of the method as well as illustrative experimental results. Several radiofrequency probe designs suitable for high field NMR imaging have been experimentally evaluated and their modification and construction are also described. The comparative results obtained indicate the merits and demerits of different designs and provide necessary guidelines for selecting the most suitable design depending on the application. Practical aspects of two- and three-dimensional imaging have been discussed and NMR images of several intact systems have been presented. Experimental methods which enable slice selection in the presence of chemically shifted species and two-dimensional chemical shift resolved imaging have "been described and illustrated using phantoms. The use of three-dimensional chemical shift resolved imaging as a potential method to map the pH and temperature distribution within an object has also been demonstrated. A preliminary investigation of the application of ³¹P NMR spectroscopy to study the biochemical transformations of the rat kidney during periods of ischemia and reperfusion has been presented. / Science, Faculty of / Chemistry, Department of / Graduate

NMR studies of paramagnetic hemoproteins have improved significantly our understanding of the structure-function relationship ofhemoproteins in general. Up to date most of the studies focus on low-spin ferric systems which are characterized by relatively narrow resonance peaks and concomitant better resolution. However, characterizing in detail the NMR spectra of high-spin ferric hemoproteins is important since there are several hemoproteins, such as peroxidases, catalases, oxygenases, and some ferricytochromes that contain high-spin iron (III) in their biologically active forms. Yet assigning resonances from heme peripheral protons and/or heme pocket residues in high-spin myoglobins is a daunting undertaking. Only a sparse number of active site residues are assigned in such instances, even for metaquo-myoglobin. The protons from the heme and heme pocket residues in high-spin complexes experience extremely fast relaxation and very broad linewidths, which impede the 2D methods that detect through-space and through-bond connectivities. It is the intention of this study to develop an effective strategy to gain more resonance assignments for fast-relaxing protons in hemoproteins. We have set out to use a combined strategy, using two-dimensional exchange spectroscopy (2D-EXSY) with two dimensional nuclear Overhauser effect spectroscopy I correlation spectroscopy I total correlation spectroscopy (NOESY/COSY/TOCSY). I demonstrate here that 2D EXSY experiments can be used to obtain assignment correlations for the heme protons of methydroxy-, metthiocyano-, metaquo-, and metimidazole-myoglobin forms. All these assignments are unambiguous and straightforward. Moreover, saturation-transfer experiments allow determination of ligand binding kinetics. Thus, the exchange rates between the metaquo- and metimidazole- or methyl substituted imidazole myoglobin complexes are estimated. The differences between the exchange rates reflect the differences in the hydrophobic and steric interactions between the ligands and the protein moiety. Although I only demonstrate the feasibility of2D EXSY for the myoglobin case, this assignment strategy should to be applicable to other hemoprotein systems.

Tin protoporphyrin IX (SnPP) is currently under investigation for the treatment of hyperbilirubinemia. The study of the complex between SnPP and equine myoglobin (EqMb) by ¹H and ¹¹⁹Sn nuclear magnetic resonance spectroscopy (NMR) can be viewed as a general model for SnPP interaction with hemoproteins. The complex formed from the equilibrium mixture of SnPP and EqMb, SnPP•EqMb, was found to have essentially the same porphyrin-binding pocket as EqMbCO and SwMbCO, including the same porphyrin orientation in the major form of the two species. ¹¹⁹Sn NMR spectroscopy was used to demonstrate that the proximal His(93)F8-metal coordination is likely to be intact in SnPP•EqMb. Minor shifts in the side chain positions of some of the residues were observed, possibly reflecting the presence of water in the sixth coordination site. SnPP•EqMb appears to be stable; it persists at room temperature for weeks and exhibits very slow exchange rates (²Hfor ¹H) for a large number of amide protons in the pH range 7-9. Events during the reconstitution of apomyoglobin (apoMb) with SnPP were probed. Thus interactions between tin(IV)protoporphyrin IX (SnPP) and equine apoMb, and between tin(IV) protoporphyrin IX dimers (SnPP)₂ and apoMb were observed by ¹H NMR and optical spectroscopic techniques. The products and intermediates observed in this situation were related to the equilibrium structure of SnPP•EqMb. Reactions of apoEqMb with SnPP and (SnPP)₂ produce different intermediates, although the final product, SnPP•EqMb, is the same for each. An intermediate observed for the reaction of SnPP with apoEqMb at pH 10 is in exchange with free SnPP, with the observed rate constant Koff ~ 1 sˉ¹; meso-proton resonances were assigned for this intermediate by correlation to SnPP resonances via chemical exchange. The intermediate observed for the reaction of (SnPP)₂ with apoEqMb at neutral pH produces another species which may be the alternate porphyrin-insertion isomer arising from a 180° rotation about the α,γ-meso axis of the porphyrin. Although optical absorbance spectroscopy of the Soret region shows evidence for the reaction of SnPP and (SnPP)₂ with apoMb, only in combination with ¹H NMR are the various processes assigned. T his study of the complex SnPP•EqMb facilitated the investigation of the more complex heme binding protein, hemopexin (Hx). Proton NMR spectroscopy is reported for the first time for the hemin complex of hemopexin, a serum protein that binds heme exceptionally tightly. Hx from cow, rat, rabbit, and human was isolated, and data for the protein were reported. Heme-bound Hx has spectral characteristics for being low-spin, paramagnetic. Deuterium isotope labels reveal the positions for the heme 1-, 3-, and 8-methyls; the 5-methyl lies in the -5 to 12 ppm region. Furthermore, two-dimensional nuclear Overhauser effect spectroscopy was used to locate other heme periphery protons, including those from the 2-vinyl and the 7-propionate. Upfield resonances are identified that are very strongly relaxed, and so are assigned to protons on the axial ligands. The information reported here contributes to the understanding of Hx as an antioxidant at the cellular level.

No description available.

No / The first report of the formation of an acetyl disulfide sialoside during the synthesis of thioglycosides is described. This compound is a by-product in the synthesis of the 2-thioacetyl sialoside commonly used in thioglycoside preparation. Our investigations into the identification of this novel disulfide are described.

No description available.

Phosphorus (³¹P) metabolites are emerging liver disease biomarkers. This work aims to develop a quantification protocol for human hepatic ³¹P magnetic resonance spectroscopy (MRS) at 7 tesla (T). It should have high SNR, deliver robust measurements of metabolite concentrations with high reproducibility, and be feasible to use in clinical studies. This will allow detailed characterization of liver metabolism in diseases such as cirrhosis, increasing the utility of ³¹P-MRS as a clinical tool. A 3D chemical shift imaging method using a 16 channel ³¹P array at 7 T is chosen to give high SNR ³¹P spectra from the human liver in vivo, while also providing good spatial localization and spectral resolution. The Oxford Spectroscopy Analysis (OXSA) toolbox, our MATLAB-based processing software package, is introduced and adaptations for analysis of liver spectra are described. Five volunteers were scanned to determine T₁s for the ten visible ³¹P metabolites. Simulations were used to determine design criteria for calibration phantoms at 1.5, 3 and 7 T. I compare three candidate approaches to give "absolute" concentrations in mmol/L wet tissue using a 10 cm loop coil, and then extend these approaches to data acquired using the 16 element receive array. The final protocol was applied to data acquired in ten healthy volunteers and eleven patients with cirrhosis to determine reproducibility and the differences between healthy and diseased livers. This protocol allows distinction between healthy and cirrhotic livers with 90% specificity and sensitivity, using cut-offs in either Î3-adenosine triphosphate or inorganic phosphate concentrations. This ³¹P-MRS absolute quantification protocol is an important first step in fully utilising the increased SNR afforded by the 7 T scanner, offering valuable insight into liver metabolism, and paving the way for other novel ³¹P-MRS methods to be developed in the liver at 7 T.

Keung Yim Mei. / "October 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 187-195). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Analysis in chemistry has always been hindered by the presence of impurities in samples or mixtures that are difficult to separate. Nuclear magnetic resonance has proven to be one of the most powerful analysis techniques to enable the study of mixtures by pseudoseparation using molecular parameters such as the diffusion coefficient through the application of the DOSY technique. In order to extend the application of this technique, an improvement has been proposed know as matrix-assisted DOSY (MAD-DOSY) or chromatographic NMR. This technique is based on the addition of a sample modifier that will interact differently with the molecules, varying and separating their diffusion coefficients, or even changing slightly the chemical shifts. To extend the application of chromatographic NMR, size exclusion stationary phases have been combined with DOSY experiments. These studies have been applied to analyze mixtures modifying the diffusion coefficient in terms of size exclusion behavior and to increase the understanding of the interactions between the analytes and the stationary phase. These studies have been published in Magnetic Resonance in Chemistry. One of the main issues when using DOSY is spectral overlapping, which is the main cause of poor resolution. In addition to this problem, a consequence of using stationary phases is the appearance of increased broadening of the signals due to differences in magnetic susceptibility. Thus, to achieve the aim, the study of diffusion properties have been performed under HR-MAS conditions which can help to remove susceptibility effect, but has complicating effects on the DOSY experiment. A method to obtain reliable diffusion measurements under HR-MAS have been developed using a D2O sample. Different conditions have been investigated including different pulse sequences, variation of parameters of the pulse sequence (diffusion delay or gradient strength), spinning rate and synchronization of the pulse sequence with the sample spinning. Also improvements in sample preparation as the addition of spacers in different locations of the sample rotor, to both reduce radial field variations and the sample volume, in order to obtain the most accurate diffusion values. This method have been published in Magnetic Resonance in Chemistry. The method have been applied to a wide range of molecules to extend the understanding of diffusion under HR-MAS conditions. In order to extend the range of application of NMR chromatography, a complementary study of the analysis of a mixture of different enantiomers including ethylenediamine cobalt complexes, aminoacids and some other organic small molecules adding to the sample a chiral stationary phase as a sample modifier is included in the final chapter of this thesis.