Novel and highly sensitive mixed-polymeric electrokinetic chromatography system for determination of contaminants and impurities of heparin samples

A mixed‐polymeric electrokinetic chromatography system has been developed for the simultaneous determination of a contaminant like oversulfated condroitin sulfate (OSCS) and impurities expressed as dermatan (Der) in heparin (Hep) samples. The EKC system consisted of 0.5% w/v polymeric β‐CD, 0.4% w/v tetronic^® 1107 and 400 mM tris‐phosphate buffer at pH 3.5. The optimized electrophoretic conditions included the use of an uncoated‐silica capillary of 50 cm of total length and 75 μm id, an applied voltage of ?7 kV, a temperature of 30°C and 200 nm UV‐detection. The highly sensitive method developed showed low values of LOD, 0.07% w/w (0.07 μg/mL) (OSCS) and 0.1% w/w (0.1 μg/mL) (Der), and values of LOQ 0.2% w/w (0.2 μg/mL) (OSCS) and 0.3% w/w (0.3 μg/mL) (Der) with a concentration level of Hep sample as low as 0.1 mg/mL. Additional parameters of validation such as specificity, linearity, accuracy, and robustness were evaluated according to international guidelines. Owing to its simplicity, high sensitivity, and reliability, the proposed method can be an advantageous alternative to the traditional methodologies for the analysis of Hep in raw material and specially in finished products because of the low amounts of Hep sample required.     

Ferroxidase Activity in Eukaryotic Ferritin is Controlled by Accessory‐Iron‐Binding Sites in the Catalytic Cavity

Ferritins are iron‐storage nanocage proteins that catalyze the oxidation of Fe²⁺ to Fe³⁺ at ferroxidase sites. By a combination of structural and spectroscopic techniques, Asp140, together with previously identified Glu57 and Glu136, is demonstrated to be an essential residue to promote the iron oxidation at the ferroxidase site. However, the presence of these three carboxylate moieties in close proximity to the catalytic centers is not essential to achieve binding of the Fe²⁺ substrate to the diferric ferroxidase sites with the same coordination geometries as in the wild‐type cages.     

Redox characteristics of a de novo quinone protein

The electrochemistry of 2,6-dimethylbenzoquinone (DMBQ) has been characterized for three different systems: DMBQ freely solvated in aqueous buffer; DMBQ bound to a neutral, blocked cysteine (N-acetyl-L-cysteine methyl ester) and the resulting DMBQ-bCys compound solvated in aqueous buffer; and DMBQ bound to a small model protein denoted alpha(3)C. The goal of this study is to detect and characterize differences in the redox properties of the protein-ligated DMBQ relative to the solvated quinones. The alpha(3)C protein used here is a tryptophan-32 to cysteine-32 variant of the structurally defined alpha(3)W de novo protein (Dai et al. J. Am. Chem. Soc. 2002, 124, 10952-10953). The properties of alpha(3)C were recently described (Hay et al. Biochemistry 2005, 44, 11891-11902). DMBQ was covalently bound to bCys and alpha(3)C through a sulfur substitution reaction with the cysteine thiol. In contrast to the solvated DMBQ and DMBQ-bCys compounds, diffusion controlled electrochemistry of DMBQ-alpha(3)C showed well-behaved and fully reversible n = 2 oxidation/reduction with a peak separation of approximately 30 mV between pH 5 and 9. DMBQ-alpha(3)C could also be immobilized on a gold electrode modified with a self-assembled monolayer of 3-mercaptopropionoic acid, allowing the measurement, by cyclic voltammetry, of an apparent rate of electron transfer of 22 s(-1). The (cysteine) sulfur substitution significantly lowers one of the hydroquinone pKA's from 10.4 in DMBQ to 6.8 in DMBQ-bCys. This pKA is slightly elevated in DMBQ-alpha(3)C to 7.0 and the E1/2 at pH 7.0 is raised by 110 mV from +190 mV in DMBQ-bCys to +297 mV in DMBQ-alpha(3)C.     

Chemical composition and volatile constituents of Anthyllis barba-jovis

Six flavonol glycosides were isolated and identified from the aerial parts of Anthyllis barba-jovis L. (Leguminosae), together with two coumarins and D-pinitol, a taxonomic marker of Leguminosae family. The structural elucidation of all compounds was based on their (1)H- and (13)C-NMR spectral data and bidimensional experiments. The total flavonoid content was also determined, according to the method described in the Italian pharmacopoeia. In order to complete the phytochemical investigation on A. barba-jovis, the essential oils from flowers and seeds were obtained by hydrodistillation and analysed by GC-MS; fresh flowers were analysed also directly by solid phase micro extraction (SPME).     

Precision of Raman depolarization and optical attenuation measurements of sound tooth enamel

The demineralization of enamel that is associated with early caries formation affects the optical properties of the enamel. Polarized Raman spectroscopy and optical coherence tomography have been used to detect these changes and potentially offer a means to detect and monitor early caries development. The total optical attenuation coefficient as measured by optical coherence tomography and the polarization anisotropy of the Raman peak arising from the symmetric ν₁ vibration of at approximately 959 cm⁻¹ have been demonstrated as being sensitive markers of early caries. This ex vivo study on extracted human teeth demonstrates that these measurements can be made with reasonable precision with concomitantly good repeatability and reproducibility in sound enamel. Such reliability is crucial for these techniques to have a practical clinical value.     

H,C, Pt NMR study on platinum(II) interaction with sulphur containing Amadori compounds

The NMR study on the interaction of Pt(II) with Amadori compounds is performed. The Amadori compounds are derived from the reaction of β-d-glucose with l-cystine leading to N,N′-di-(1-deoxy-β-fructos-1-yl)-l-cystine [FruCyscys], and with l-methionine leading to N-(1-deoxy-β-fructos-1-yl)-l-methionine [FruMet].     

Studies on simulated ageing of paper by photochemical degradation

The study of the ageing of two paper types was performed on monitoring it during a simulated process by means of the measure of the photocatalytic degradation of the paper cellulose. Such evaluation was possible due to the combined responses of a photosensor based on titanium dioxide in suspension, of an enzymatic biosensor based on superoxide dismutase (SOD), of a specific conductivity sensor and of an enzymatic biosensor based on glucose oxidase.     

Dynamical Aspects of TEM-1 β-Lactamase Probed by Molecular Dynamics

The dynamical aspects of the fully hydrated TEM-1 β-lactamase have been determined by a 5 ns Molecular Dynamics simulation. Starting from the crystallographic coordinates, the protein shows a relaxation in water with an overall root mean square deviation from the crystal structure increasing up to 0.17 nm, within the first nanosecond. Then a plateau is reached and the molecule fluctuates around an equilibrium conformation. The results obtained in the first nanosecond are in agreement with those of a previous simulation (Diaz et al., J. Am. Chem. Soc., (2003) 125, 672–684). The successive equilibrium conformation in solution shows an increased mobility characterized by the following aspects. A flap-like translational motion anchores the Ω-loop to the body of the enzyme. A relevant part of the backbone dynamics implies a rotational motion of one domain relative to the other. The water molecules in the active site can exchange with different residence times. The H-bonding networks formed by the catalytic residues are frequently interrupted by water molecules that could favour proton transfer reactions. An additional simulation, where the aspartyl dyad D214–D233 was considered fully deprotonated, shows that the active site is destabilized.     

A polystyrene-supported triflating reagent for the synthesis of aryl triflates

An insoluble polystyrene-supported triflating reagent has been prepared by suspension co-polymerization of N-(4-vinylphenyl)trifluoromethanesulphonimide, styrene and the JandaJel^® cross-linker. This reagent, in the presence of triethylamine, allows for the efficient synthesis of aryl triflates from a wide range of phenols in a process that permits the desired product to be isolated from the reaction mixture in essentially pure form via several filtration and concentration operations. Adding to the utility of this reagent is its ability to be easily recovered, regenerated and reused. Both soluble and insoluble bifunctional polymers containing trialkylamine moieties in addition to triflimide groups were also prepared and examined as triflating reagents. Unfortunately these reagents afforded only modest yields of the desired products in representative reactions.