Time-resolved enzymatic determination of glucose using a fluorescent europium probe for hydrogen peroxide

An enzymatic assay for glucose based on the use of the fluorescent probe for hydrogen peroxide, europium(III) tetracycline (EuTc), is described. The weakly fluorescent EuTc and enzymatically generated H₂O₂ form a strongly fluorescent complex (EuTc–H₂O₂) whose fluorescence decay profile is significantly different. Since the decay time of EuTc–H₂O₂ is in the microseconds time domain, fluorescence can be detected in the time-resolved mode, thus enabling substantial reduction of background fluorescence. The scheme represents the first H₂O₂-based time-resolved fluorescence assay for glucose not requiring the presence of a peroxidase. The time-resolved assay (with a delay time of 60 s and using endpoint detection) enables glucose to be determined at levels as low as 2.2 mol L^–1, with a dynamic range of 2.2–100 mol L^–1. The method also was adapted to a kinetic assay in order to cover higher glucose levels (mmol L^–1 range). The latter was validated by analyzing spiked serum samples and gave a good linear relationship for glucose levels from 2.5 to 55.5 mmol L^–1. Noteworthy features of the assay include easy accessibility of the probe, large Stokes shift, a line-like fluorescence peaking at 616 nm, stability towards oxygen, a working pH of approximately 7, and its suitability for both kinetic and endpoint determination.     

Interactions of Some Amino Acids with Aqueous Tetraethylammonium Bromide at 298.15 K: A Volumetric Approach

The apparent molar volumes, V_,2, of glycine, L-alanine, DL--amino-n-butyric acid, L-valine, and L-leucine have been determined in aqueous 0.25, 0.75, 1.0, and 1.5 mol-dm^–3 tetraethylammonium bromide (TEAB) solutions by density measurements at 298.15 K. These data have been used to calculate the infinite dilution apparent molar volumes, V_2,m, for the amino acids in aqueous tetraethylammonium bromide and the standard partial molar volumes of transfer (_tr V_2,m) of the amino acids from water to the aqueous salt solutions. The linear correlation of V_2,m for a homologous series of amino acids has been utilized to calculate the contribution of the charged end groups (NH₃⁺, COO^–), CH₂ group, and other alkyl chains of the amino acids to V_2,m. The results of the standard partial molar volumes of transfer from water to aqueous tetraethylammonium bromide have been interpreted in terms of ion–ion, ion–polar, and hydrophobic–hydrophobic group interactions. The volume of transfer data suggest that ion–ion or ion–hydrophilic interactions are predominant in the case of glycine and alanine, and hydrophobic–hydrophobic group interactions are predominant in the case of DL--amino butyric acid, L-valine, and L-leucine.     

Parameterization of peptide 13C carbonyl chemical shielding anisotropy in molecular dynamics simulations.

NMR chemical shielding anisotropy (CSA) relaxation is an important tool in the study of dynamical processes in proteins and nucleic acids in solution. Herein, we investigate how dynamical variations in local geometry affect the chemical shielding anisotropy relaxation of the carbonyl carbon nucleus, using the following protocol: 1) Using density functional theory, the carbonyl (13)C' CSA is computed for 103 conformations of the model peptide group N-methylacetamide (NMA). 2) The variations in computed (13)C' CSA parameters are fitted against quadratic hypersurfaces containing cross terms between the variables. 3) The predictive quality of the CSA hypersurfaces is validated by comparing the predicted and de novo calculated (13)C' CSAs for 20 molecular dynamics snapshots. 4) The CSA fluctuations and their autocorrelation and cross correlation functions due to bond-length and bond-angle distortions are predicted for a chemistry Harvard molecular mechanics (CHARMM) molecular dynamics trajectory of Ca(2+)-saturated calmodulin and GB3 from the hypersurfaces, as well as for a molecular dynamics (MD) simulation of an NMA trimer using a quantum mechanically correct forcefield. We find that the fluctuations can be represented by a 0.93 scaling factor of the CSA tensor for both R(1) and R(2) relaxations for residues in helix, coil, and sheet alike. This result is important, as it establishes that (13)C' relaxation is a valid tool for measurement of interesting dynamical events in proteins.     

Structural assignment of organotin hydrides containing the oxazoline ligand

A series of triorganotin hydrides and diorganotin dihydrides containing the optically active 2-(4-isopropyl-2-oxazolinyl)-5-phenyl ligand have been characterized by means of the multinuclear low-temperature NMR investigations, the results of which are discussed. In the corresponding organotin hydrides values of the ¹J(¹H-^117/119Sn) couplings appeared to be temperature dependent, supporting an axial/equatorial position of the hydrogen attached to the tin.     

Tri-component diblock copolymers of poly(ethylene glycol)–poly(ε-caprolactone-<Emphasis Type="Italic">co</Emphasis>-lactide): synthesis,characterization and loading camptothecin

Biodegradable tri-component diblock copolymer was synthesized by bulk copolymerization of ε-caprolactone (CL) and D, L-lactide (LA) in the presence of methoxy poly(ethylene glycol) (MePEG), using stannous octoate as catalyst. Their chemical structure and physical properties were investigated by GPC, NMR, DSC, TGAand XRD. The increase of CL/LA ratio in the diblock copolymer leads to lower T _g, higher decomposition temperature and crystallinity. Nanoparticles formulated from MePEG–poly(CL-co-LA) (PCAE) possess spherical structure, which was characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The DLS results indicate that the particle size increased with the increase of CL/LA ratio and the hydrophobic fragment length in the copolymer. The drug encapsulation efficiency and the drug release behavior in vitro conditions of camptothecin were measured by high performance liquid chromatography (HPLC). The encapsulation efficiency can be achieved as high as 84.4% and the release behavior can be made well-controlled. MePEG–poly(CL-co-LA) nanoparticles might have a great potential as carriers for hydrophobic drugs.     

Five-coordinate Cobalt(II), Nickel(II) and Zinc(II) Complexes Derived from 2-pyridine-2-yl-3-(pyridine-2-carboxylideneamino)-1,2-dihydroquinazolin-4(3H)-one. The Crystal Structure of the Cobalt(II) Complex

The synthesis of cobalt(II), nickel(II) and zinc(II) complexes of 2-pyridine-2-yl-3(pyridine-2-carboxylideneamino)-1,2-dihydroquinazolin-4(3H)-one is described. The ligand and metal complexes were characterized by elemental analysis, conductivity measurements, spectral (u.v.–vis., i.r., 1D n.m.r., 2D hetcor and mass) and thermal studies. The cobalt(II) complex crystallizes as pink crystals in the monoclinic crystal system, space group P21/n with a = 10.066(6) Å, b = 15.929(9) Å, c = 12.624(7) Å, α = 90.00(9)°, β = 110.850 (8)°, γ = 90.00, V = 1891.5 (18) ų and Z = 4. The geometry around the cobalt atom is distorted trigonal bipyramidal with τ = 0.83 [structural parameter, τ = (β − α)/60; where α and β are the two basal angles in a five coordinate complex].     

AFM of adsorbed polyelectrolytes on cellulose I surfaces spin-coated on silicon wafers

Thin layers of cellulose I nanocrystals were spin-coated onto silicon wafers to give a flat model cellulose surface. A mild heat treatment was required to stabilize the cellulose layer. Interactions of this surface with polyelectrolyte layers and multilayers were probed by atomic force microscopy in water and dilute salt solutions. Deflection–distance curves for standard silicon nitride tips were measured for silicon, cellulose-coated silicon, and for polyelectrolytes adsorbed on the cellulose surface. Transfer of polymer to the tip was checked by running deflection–distance curves against clean silicon. Deflection–distance curves were relatively insensitive to adsorbed polyelectrolyte, but salt addition caused transfer of cationic polyelectrolyte to the tip, and swelling of the polyelectrolyte multilayers.     

What to Learn from a Comparative Genomic Sequence Analysis of <Emphasis Type="Italic">L</Emphasis>-Carnitine Dehydrogenase

Summary. In contrast to eukaryotic cells certain eubacterial strains have acquired the ability to utilize L-carnitine (R-(–)-3-hydroxy-4-(trimethylamino)butyrate) as sole source of energy, carbon and nitrogen. The first step of the L-carnitine degradation to glycine betaine is catalysed by L-carnitine dehydrogenase (L-CDH, EC 1.1.1.108) and results in the formation of the dehydrocarnitine. During the oxidation of L-carnitine a simultaneous conversion of the cofactor NAD⁺ to NADH takes place. This catabolic reaction has always been of keen interest, because it can be exploited for spectroscopic L-carnitine determination in biological fluids – a quantification method, which is developed in our lab – as well as L-carnitine production.Based on a cloned L-CDH sequence an expedition through the currently available prokaryotic genomic sequence space began to mine relevant information about bacterial L-carnitine metabolism hidden in the enormous amount of data stored in public sequence databases. Thus by means of homology-based and context-based protein function prediction is revealed that L-CDH exists in certain eubacterial genomes either as a protein of approximately 35 kDa or as a homologous fusion protein of approximately 54 kDa with an additional putative domain, which is predicted to possess a thioesterase activity. These two variants of the enzyme are found on one hand in the genome sequence of bacterial species, which were previously reported to decompose L-carnitine, and on the other hand in gram-positive bacteria, which were not known to express L-CDH. Furthermore we could not only discover that L-CDH is located in a conserved genetic entity, which genes are very likely involved in this L-carnitine catabolic pathway, but also pinpoint the exact genomic sequence position of several other enzymes, which play an essential role in the bacterial metabolism of L-carnitine precursors.     

Limiting Partial Molar Volumes of Electrolytes in 2-Methyl-2-Butanol <Emphasis Type="Bold">+</Emphasis> Water Mixtures at 298.15 K

Partial molar volumes at infinite dilution, V⁰₂, of alkali–metal halides (LiCl, NaCl KCl RbCl CsCl, NaBr, KBr, KI), tetra-n-alkylammonium bromides, R₄NBr (R=Me, Et, n-Pr, n-Bu, n-Pen), NaBPh₄, and Ph₄PCl have been determined in binary solvent mixtures of water with 2-methyl-2-butanol covering the water-rich region and the alcohol-rich region at 298.15 K. V⁰₂ for alkali–metal halides show relatively little dependence on the solvent composition. However, in the case of hydrophobic electrolytes the observed effects are more pronounced. A good linear dependence between V⁰₂(R₄NBr) and the molecular weight of the tetra-n-alkylammonium cation is found. Limiting single-ion volumes have been obtained using the assumption that V⁰(Ph₄P⁺)–V⁰(BPh^–₄)=2.0 cm³-mol^–1. The trends in the single-ion volumes are discussed in both solvent regions.     

Three-dimensional structural features and the toxicity of aminobenzenes and phenols

There are many organic pollutants in the environment, such as polychlorinated biphenyl, polycyclic aromatic hydrocarbons, dichlorodiphenyl-trichloroethane (DDT), and polychlorinated naphthalene. These organic pollutants are persistent,liposoluble and easily cumulated in organism; consequently, the potential toxicity will be high. Risk assessment of industrial chemicals is currently carried out using scanty experimental data, because many of these chemicals have very little or no test data. S…