1,721,051 research outputs found
Anomerisation of fluorinated sugars by mutarotase studied by 19F NMR two-dimensional exchange spectroscopy
No full textFive
19F-substituted glucose analogues were used to probe the activity and mechanism of the enzyme mutarotase by using magnetisation-exchange NMR spectroscopy. The sugars (2-fluoro-2-deoxy-d-glucose, FDG2; 3-fluoro-3-deoxy-d-glucose, FDG3; 4-fluoro-4-deoxy-d-glucose, FDG4; 2,3-difluoro-2,3-dideoxy-d-glucose, FDG23; and 2,2,3,3-tetrafluoro-2,3-dideoxy-d-glucose (2,3-dideoxy-2,2,3,3-tetrafluoro-d-erythro-hexopyranose), FDG2233) showed separate
19F NMR spectroscopic resonances from their respective α- and β-anomers, thus allowing two-dimensional exchange spectroscopy measurements of the anomeric interconversion at equilibrium, on the time scale of a few seconds. Mutarotase catalysed the rapid exchange between the anomers of FDG4, but not the other four sugars. This finding, combined with previous work identifying the mechanism of the anomerisation by mutarotase, suggests that the rotation around the C1-C2 bond of the pyranose ring is the rate-limiting reaction step. In addition to d-glucose itself, it was shown that all other fluorinated sugars inhibited the FDG4 anomerisation, with the tetrafluorinated FDG2233 being the most potent inhibitor. Inhibition of mutarotase by F-sugars paves the way for the development of novel fluorinated compounds that are able to affect the activity of this enzyme in vitro and in vivo.
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Quantitative analysis of 2D EXSY NMR spectra of strongly coupled spin systems in transmembrane exchange
No full textSolute translocation by membrane transport proteins is a vital biological process that can be tracked, on the sub-second timescale, using nuclear magnetic resonance (NMR). Fluorinated substrate analogues facilitate such studies because of high sensitivity of 19F NMR and absence of background signals. Accurate extraction of translocation rate constants requires precise quantification of NMR signal intensities. This becomes complicated in the presence of J-couplings, cross-correlations, and nuclear Overhauser effects (NOE) that alter signal integrals through mechanisms unrelated to translocation. Geminal difluorinated motifs introduce strong and hard-to-quantify contributions from non-exchange effects, the nuanced nature of which makes them hard to integrate into data analysis methodologies. With analytical expressions not being available, numerical least squares fitting of theoretical models to 2D spectra emerges as the preferred quantification approach. For large spin systems with simultaneous coherent evolution, cross-relaxation, cross-correlation, conformational exchange, and membrane translocation between compartments with different viscosities, the only available simulation framework is Spinach. In this study, we demonstrate GLUT-1 dependent membrane transport of two model sugars featuring CF2 and CF2CF2 fluorination motifs, with precise determination of translocation rate constants enabled by numerical fitting of 2D EXSY spectra. For spin systems and kinetic networks of this complexity, this was not previously tractable.</p
Transmembrane exchange of fluorosugars: Characterization of red cell GLUT1 kinetics using <sup>19</sup>F NMR
No full textWe have developed a new approach, to our knowledge, to quantify the equilibrium exchange kinetics of carrier-mediated transmembrane transport of fluorinated substrates. The method is based on adapted kinetic theory that describes the concentration dependence of the transmembrane exchange rates of two competing, simultaneously transported species. Using the new approach, we quantified the kinetics of membrane transport of both anomers of three monofluorinated glucose analogs in human erythrocytes (red blood cells) using 19F NMR exchange spectroscopy. An inosine-based glucose-free medium was shown to promote survival and stable metabolism of red blood cells over the duration of the experiments (several hours). Earlier NMR studies only yielded the apparent rate constants and transmembrane fluxes of the anomeric species, whereas we could categorize the two anomers in terms of the catalytic activity (specificity constants) of the glucose transport protein GLUT1 toward them. Differences in the membrane permeability of the three glucose analogs were qualitatively interpreted in terms of local perturbations in the bonding of substrates to key amino acid residues in the active site of GLUT1. The methodology of this work will be applicable to studies of other carrier-mediated membrane transport processes, especially those with competition between simultaneously transported species. The GLUT1-specific results can be applied to the design of probes of glucose transport or inhibitors of glucose metabolism in cells, including those exhibiting the Warburg effect.</p
NMR of 133Cs+ in stretched hydrogels: one-dimensional, z- and NOESY spectra, and probing the ion’s environment in erythrocytes
No full text(133)Cs nuclear magnetic resonance (NMR) spectroscopy was conducted on (133)Cs(+) in gelatin hydrogels that were either relaxed or stretched. Stretching generated a septet from this spin-7/2 nucleus, and its nuclear magnetic relaxation was studied via z-spectra, and two-dimensional nuclear Overhauser (NOESY) spectroscopy. Various spectral features were well simulated by using Mathematica and the software package SpinDynamica. Spectra of CsCl in suspensions of human erythrocytes embedded in gelatin gel showed separation of the resonances from the cation inside and outside the cells. Upon stretching the sample, the extracellular (133)Cs(+) signal split into a septet, while the intracellular peak was unchanged, revealing different alignment/ordering properties of the environment inside and around the cells. Differential interference contrast light microscopy confirmed that the cells were stretched when the overall sample was elongated. Analysis of the various spectral features of (133)Cs(+) reported here opens up applications of this K(+) congener for studies of cation-handling by metabolically-active cells and tissues in aligned states
PFG NMR diffusion experiments for complex systems
No full textMany practical applications of diffusion NMR, ranging from biomedical to industrial, entail the measurement of low-concentration solutes in non-deuterated, compositionally complex systems. The aim of this article is to present examples of robust, versatile diffusion experiments that can be used with non-deuterated solvents and at non-ambient temperatures. Specifically, three experiments are presented in detail: CONVEX, which combines excitation-sculpting solvent suppression with double-echo convection-compensating PGSE; DQDiff, which implements double-quantum filtered diffusion measurements in a convection-compensating mode; and applications of Oscillating-Gradient Spin-Echo (OGSE) to systems with homonuclear scalar couplings. These examples are based on the recent work by the authors and relate to a variety of systems, ranging from simple solutions to colloidal and polymeric systems. Besides the applied aspects, we review the general methodology used to treat the effects of diffusion and flow in NMR experiments, and apply this theory to derive the diffusion attenuation expression for each of the experiments presented. The paper should be useful to beginners as well as advanced users of general NMR wishing to learn about diffusion measurements
Erratum: Pulsed field gradient nuclear magnetic resonance as a tool for studying drug delivery systems
No full textReview of mutarotase in ‘Metabolic Subculture’ and analytical biochemistry: prelude to 19F NMR studies of its substrate specificity and mechanism
No full textThis is the first paper in a sequential pair devoted to the enzyme mutarotase (aldose 1-epimerase; EC 5.1.3.3). Here, the broader context of the physiological role of mutarotase, among those enzymes considered to be part of 'metabolic structure', is reviewed. We also summarise the current knowledge about the molecular mechanism and substrate specificity of the enzyme, which is considered in the context of the binding of fluorinated glucose analogues to the enzyme's active site. This was done as a prelude to our experimental studies of the anomerisation of fluorinated sugars by mutarotase that are described in the following paper.</p
Convection-compensating diffusion experiments with phase-sensitive double-quantum filtering
No full textWe present a design scheme for phase-sensitive, convection-compensating diffusion experiments with gradient-selected homonuclear double-quantum filtering. The scheme consists of three blocks: a 1/2J evolution period during which antiphase single-quantum coherences are created; a period of double-quantum evolution; and another 1/2J period, during which antiphase single-quantum coherences are converted back into an in-phase state. A single coherence transfer pathway is selected using an asymmetric set of gradient pulses, and both diffusion sensitization and convection compensation are built into the gradient coherence transfer pathway selection. Double-quantum filtering can be used either for solvent suppression or spectral editing, and we demonstrate examples of both applications. The new experiment performs well in the absence of a field-frequency lock and does not require magnitude Fourier transformation. The proposed scheme may offer advantages in diffusion measurements of spectrally crowded systems, particularly small molecules solubilized in colloidal solutions or bound to macromolecules
Sub-minute kinetics of human red cell fumarase: 1H spin-echo NMR spectroscopy and 13C rapid-dissolution dynamic nuclear polarization
No full textFumarate is an important probe of metabolism in hyperpolarized magnetic resonance imaging and spectroscopy. It is used to detect the release of fumarase in cancer tissues, which is associated with necrosis and drug treatment. Nevertheless, there are limited reports describing the detailed kinetic studies of this enzyme in various cells and tissues. Thus, we aimed to evaluate the sub-minute kinetics of human red blood cell fumarase using nuclear magnetic resonance (NMR) spectroscopy, and to provide a quantitative description of the enzyme that is relevant to the use of fumarate as a probe of cell rupture. The fumarase reaction was studied using time courses of 1H spin-echo and 13C-NMR spectra. 1H-NMR experiments showed that the fumarase reaction in hemolysates is sufficiently rapid to make its kinetics amenable to study in a period of approximately 3 min, a timescale characteristic of hyperpolarized 13C-NMR spectroscopy. The rapid-dissolution dynamic nuclear polarization (RD-DNP) technique was used to hyperpolarize [1,4-13C]fumarate, which was injected into concentrated hemolysates. The kinetic data were analyzed using recently developed FmRα analysis and modeling of the enzymatic reaction using Michaelis–Menten equations. In RD-DNP experiments, the decline in the 13C-NMR signal from fumarate, and the concurrent rise and fall of that from malate, were captured with high spectral resolution and signal-to-noise ratio, which allowed the robust quantification of fumarase kinetics. The kinetic parameters obtained indicate the potential contribution of hemolysis to the overall rate of the fumarase reaction when 13C-NMR RD-DNP is used to detect necrosis in animal models of implanted tumors. The analytical procedures developed will be applicable to studies of other rapid enzymatic reactions using conventional and hyperpolarized substrate NMR spectroscopy
NMR Study of the Association of Propofol with Nonionic Surfactants
No full textThe general anesthetic 2,6-diisopropylphenol (propofol) is very poorly soluble in water and is normally administered in the form of an emulsion. We demonstrated that several commercially available nonionic surfactants (Tween 80, Cremophor EL, Poloxamer 188, Poloxamer 407, Solutol HS15, and Vitamin E TPGS)render propofol soluble with a specific solubilization capacity of at least 0.1 g/g. The room-temperature stability of the solutions appeared to be limited only by the chemical stability of the compounds involved. Theassociation between propofolandthe surfactantswasinvestigated by various NMR approaches, including measurements of diffusion coefficients, 1H longitudinal relaxation times, and the magnitude of intermolecular nuclear Overhauser effects. The results were consistent with the micellar solubilization mechanism of propofol by the surfactants (unimer solubilization in the case of Poloxamer 188). The 1H longitudinal relaxation and diffusion behavior of propofol were monoexponential in each case. Solubilization caused a considerable shortening of propofol’s proton T1’s. The values of the diffusion coefficient of propofol were several percent higher than those of surfactants. This was explained by the partitioning of propofol between swollen micelles and the aqueous solution. Diffusion measurements also revealed the presence of a rapidly diffusing ethylene oxide population in surfactant solutions, which is consistent with free poly(ethylene oxide) (PEO) known to be present in commercially produced surfactants. The free PEO blocks exhibited molecular association with the extramicellar propofol
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