Cross-correlated relaxation enhanced 1H[bond]13C NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes

A comparison of HSQC and HMQC pulse schemes for recording (1)H[bond](13)C correlation maps of protonated methyl groups in highly deuterated proteins is presented. It is shown that HMQC correlation maps can be as much as a factor of 3 more sensitive than their HSQC counterparts and that the sensitivity gains result from a TROSY effect that involves cancellation of intra-methyl dipolar relaxation interactions. (1)H[bond](13)C correlation spectra are recorded on U-[(15)N,(2)H], Ile delta 1-[(13)C,(1)H] samples of (i) malate synthase G, a 723 residue protein, at 37 and 5 degrees C, and of (ii) the protease ClpP, comprising 14 identical subunits, each with 193 residues (305 kDa), at 5 degrees C. The high quality of HMQC spectra obtained in short measuring times strongly suggests that methyl groups will be useful probes of structure and dynamics in supramolecular complexes.     

Allylic interactions in organometallics: probing Electronic structure in (η5-C5H5)Fe(CO)2R,R = CH3, η1-C3H5, η1-C5H5.

The He(I) photoelectron spectra of (η⁵-C₅H₅)Fe(CO)₂R, where R = CH₃, η¹-C₃H₅ and η¹-C₅H₅, have been recorded. The lowest lying ion states result from ionization of molecular orbitals with large Fe 3d character; these move to lower anergy when R places double bonds in an allylic relationship to the metal atom. The cyclic voltammetric oxidation potential correlates well with the energies of the lowest ion states. A significant interaction between olefin π orbitals and the allylic metal center is proposed.     

On an estimate for Bloch and Doob norm and covering problem in Doob's class

For any fixed 0 < π ? 2π, let D(π) be the family of all holomorphic functions in the unit disk Δ which satisfy (i)f(0) = 0 and (ii) $\text{lim inf}{}_{\mathrm{z\; \to \; \pi ¦f(z)¦\; ?\; 1}}$, for all π lying on some arc A_f ? ?Δ with arclength $\text{¦A}{}_{\mathrm{f}}\text{¦ ? π}$. We show that for each 0 < ε < 1, there is a π₀ > 0 such that for any f?D(π) with π < π₀, the Bloch and Doob norm respectively satisfy

$\text{6f6}{}_{\mathrm{B}}\text{=}\text{sup}\text{z?Δ}\text{|f′(z)| (1?|z|}{}^2\text{) > 2(1 ? ε)}\text{log}\text{1+}\text{cos}\text{(}\text{p}\text{2}\text{1?}\text{cos}\text{(}\text{p}\text{2}{}^{\mathrm{?1}}$

$\text{6f6}{}_{\mathrm{D}}\text{=}\text{sup}\text{z?Δ}\text{|f′(z)| (1?|z|) > (1 ? ε)}\text{log}\text{1}\text{1?}\text{cos}\text{(}\text{p}\text{2}{}^{\mathrm{?1}}$

These two estimates do not hold with ε = 0.     

Synthesis and Molecular Recognition of Molecularly Imprinted Polymer with Ibuprofen as Template

Ibuprofen and ketoprofen are chemically similar non‐steroidal anti‐inflammatory drugs widely used in the treatment of arthritis. Using a molecular imprinting technique, a simple and rapid method was developed for the simultaneous separation and determination of ibuprofen and ketoprofen. Molecular imprinting introduces artificial binding sites into a synthetic polymer matrix, allowing it to exhibit selective rebinding of template molecules. Imprinted polymers can be regarded as an HPLC stationary phase, important for pharmaceutical analysis. Most molecularly imprinted polymers (MIPs) are synthesized by free radical polymerization of functional monomers, resulting in an excess of crosslinking monomers. In this study, MIPs have been prepared with a ibuprofen template, which can form intramolecular hydrogen bonds. Methacrylic acid (MAA) and ethyleneglycol dimethacrylate (EGDMA) were used as the functional monomer and cross‐linker, respectively. Bulk polymerization was carried out at 4 °C under UV radiation. The resulting MIP was ground into 25?44 μm particles, which were slurry‐packed into analytical columns. Template molecules were removed by methanol‐acetic acid (9:1, v/v). We evaluated the template binding performance of the MIP using HPLC, with ultraviolet (UV) detection at 234 nm. Chromatographic resolution of ibuprofen and ketoprofen on the MIPs were appraised using buffer/acetonitrile (45/55, v/v) as the mobile phase. Results show that the MIPs prepared using ibuprofen as the template had a significant molecular imprinting effect. The method was successfully applied to the separation and analysis of ibuprofen and ketoprofen in pharmaceuticals.     

Design and Construction of a Pulsed Field-Gradient NMR Probe for a High-Field Superconducting Magnet

We describe the design and construction of a high-resolution pulsed field-gradient NMR probe for use in a high-field superconducting magnet. Overview and details of the design and construction of the probe case, rf circuitry, temperature control and shielded gradient coils for a heteronuclear probe suitable for a spectrometer (Bruker MSL-300) equipped with a wide bore (i.e., 89 mm) magnet (Oxford) are given. The interior of the probe is designed so mat it may be easily moved to variable height so as to accept easily various gradient coils. A design for a novel jig for the winding of saddle-geometry rf coils is given.     

Electron spin resonance for quantitative assay of chlorpromazine in drug formulations by oxidation with cerium(IV) in sulfuric acid media

For the first time electron spin resonance (ESR) spectroscopic technique has been used for a highly selective determination of chlorpromazine. The method is based on the oxidation reaction of chlorpromazine with cerium(IV) in sulfuric acid media. In this method 3.8x10(-3) mol dm(-3) cerium(IV) was used in 0.035 mol dm(-3) sulfuric acid with the ESR spectra recorded at room temperature. A calibration equation of the following form was obtained over the linear concentration range of 10-100 ppm with a correlation coefficient (r) of 0.999: A=1.355+0.0382C. The results obtained by the ESR method were found to be comparable with those obtained by the British Pharmacopoeia (BP) method. The method suffers no interferences excipients rendering the method suitable for determination of this drug in pharmaceutical preparations.     

Chromatographic characteristics of cholesterol-imprinted polymers prepared by covalent and non-covalent imprinting methods

Cholesterol-imprinted polymers were prepared in bulk polymerization by the methods of covalent and non-covalent imprinting. The former involved the use of a template-containing monomer, cholesteryl (4-vinyl)phenyl carbonate, while the latter used the complexes of template and functional monomer, methacrylic acid or 4-vinylpyridine prior to polymerization. Columns packed with these molecularly imprinted polymers (MIPs) were all able to separate cholesterol from other steroids. For different combinations of cholesterol and beta-estradiol concentrations in a total of 1 g/l, the peak retention times for both compounds were nearly constant. The adsorption capacity for cholesterol onto the MIPs was found to significantly depend on the use of functional monomers, but the selectivity factors were only slightly different from each other at 2.9 to 3.2 since the separation was all based on the specific binding of cholesterol to recognition sites formed on the imprinted polymers. The capacity factors for cholesterol were determined to be 3.5, 4.0 and 3.1, respectively, for covalently imprinted, 4-vinylpyridine-based, and methacrylic acid-based non-covalently imprinted polymers. However, the covalently imprinted polymer was found to have a higher adsorption capacity for cholesterol and about fivefold higher chromatographic efficiency for cholesterol separation, in comparison with non-covalently imprinted polymers. The use of covalent imprinting significantly reduced the peak broadening and tailing. This advantage along with constant retention suggests that the covalently imprinted polymer has potential for quantitative analysis.     

Heterogeneous molecular distribution in supported multicomponent lipid bilayers

Membrane domains contribute important structural and functional attributes to biological membranes. We describe the heterogeneous nanoscale distribution of lipid molecules within microscale membrane domains in multicomponent lipid bilayers composed of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol). The lipids were labeled with the fluorescent lipid analogues Bodipy-PC and DiI-C20:0 to identify the distribution of individual membrane components. We used a near-field scanning optical microscope (NSOM) at room temperature to identify the nanoscale structures in the membrane. Simultaneous multicolor NSOM imaging at the emission maxima of the fluorescent analogues revealed a patchy distribution of Bodipy-PC and DiI-C20:0 indicative of phase separations in the bilayer. In a cholesterol-free system (DPPC/DLPC = 1:1), NSOM images proved that the two phosphatidylcholine molecules can coexist in domains at the micrometer level but form nanoscopic patches within the domains; DPPC occurs at the edge of the domains, whereas DLPC is present throughout the domains. In the presence of cholesterol (DPPC/DLPC = 7:3, chol = 18.9%), the two lipid molecules were more miscible but incomplete phase separations also occurred. The average domain sizes were 140-200 nm, well below the resolution capabilities of diffraction-limited light microscopy techniques; the domains were unresolvable by confocal microscopy. Our high-resolution NSOM studies of membrane domain behavior provide a better understanding of complex membrane phase phenomena in multicomponent biological membranes.     

Effect of solvent upon competitive liquid-liquid extraction of alkali metal cations by isomeric dibenzo-16-crown-5-oxyacetic acids

The selectivity and efficiency of competitive liquid-liquid extraction of alkali metal cations into organic solvents containingsym-(octyl)dibenzo-16-crown-5-oxyacetic acid (2) andsym-bis[4(5)-tert-butylbenzo]-16-crown-5-oxyacetic acid (3) have been determined. Solvents examined include: dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, 1,1,1-trichloroethane, benzene, toluene,p-xylene, chlorobenzene, 1,2-dichlorobenzene, and 1,2,3,4-tetrahydronaphthalene. The Na⁺/K⁺ and Na⁺/Li⁺ extraction ratios are highest in chloroform. The extraction selectivity is found to correlate with the diluent parameter (DP) of the organic solvent.This paper is dedicated to the memory of the late Dr C. J. Pedersen.