Glycosylation site analysis of human alpha-1-acid glycoprotein (AGP) by capillary liquid chromatography-electrospray mass spectrometry

A new anionic surfactant (RapiGest SF) was successfully used for site-specific analysis of glycosylation in human alpha-1-acid glycoprotein (AGP). By means of this analytical approach combined with capillary HPLC-mass spectrometry (and tandem mass spectrometry), the N-linked glycosylation pattern of AGP was explored. On the basis of mass matching and MS/MS experiments ca 80 different AGP-derived glycopeptides were identified. Glycosylation shows a markedly different pattern for the various glycosylation sites. At sites I and II, triantennary complex-type oligosaccharides predominate and at sites III, IV and V, tetra-antennary complex-type oligosaccharides predominate. Sites IV and V show the presence of additional N-acetyl lactosamine (Gal-GlcNAc) units (even higher degree of branching and/or longer antennae are also present).     

Rapid trace analysis of alachlor in water and vegetable samples

The use of a rapid and specific (cross-reactivity<4%) enzyme-linked immunosorbent assay (ELISA) for the determination of alachlor residues in water and vegetable samples is addressed. The analytical method consists of a fast extraction procedure followed by an optimised ELISA. The detection limit was 0.44 microg l(-1), with a linear range from 0.89 to 143.2 microg l(-1). For alachlor extraction from water samples, different solid-phase cartridges (C, Ph, C8 and C2) were assayed using MeOH as eluent. Extracts were diluted (1:4) with distilled water before ELISA. This procedure gave recoveries close to 100% with RSDs<14%. For vegetable samples, alachlor was extracted directly with MeOH and the extracts diluted 1:40 (v/v) with saline buffer prior to ELISA. The results obtained by the proposed procedure correlate well with the reference method (multiresidue extraction-GC-MS) for vegetable samples (r>0.85).     

The direct detection of a hydrogen bond in the solid state by NMR through the observation of a hydrogen-bond mediated (15)N [bond] (15)N J coupling

A new method for detecting hydrogen bonds in the solid state is presented. Using two-dimensional NMR correlation experiments, it is shown that a hydrogen-bond mediated J coupling can be observed in a powder under magic-angle spinning conditions, even though the J coupling is 2 orders of magnitude smaller than the dominant anisotropic interactions encountered in solid-state NMR. Specifically, the observation of a pair of peaks in a two-dimensional 15N-15N solid-state INADEQUATE experiment due to two nitrogens that have no covalent connectivity is attributed to the presence of a J coupling across a linking hydrogen bond.     

Photoinduced electroreduction of chlorophyllide on alkanethiol-coated mercury

Monolayers of n-alkanethiols of chain length from C12 to C18 were self-assembled on a hanging mercury drop electrode, and a film of chlorophyllide (Chlide) was adsorbed on top of them. The reduction photocurrents following illumination of the Chlide film were measured over the potential range in which the Chlide is electroinactive in the dark, and their action spectra were determined. Plotting the derivative of the photocurrents with respect to the applied potential against potential yields bell-shaped curves that can be fitted to a Gaussian. The potential of the Gaussian maximum was used to determine the reorganization energy lambda for the Chlide electroreduction process. An increase in the thiol chain length causes lambda to decrease regularly and the photocurrent to decay exponentially with the monolayer thickness, with a decay constant beta of about 0.17 A(-1).     

Purification of human leucocyte pyruvate kinase

The M2 form of pyruvate kinase (M2-PK) was purified from human leucocytes by a new method involving a succession of two different Dyematrex agarose chromatographies. The main step consisted of an orange dye affinity column with elution by fructose-1,6-diphosphate. This purification procedure allowed us to obtain M2-PK with a specific activity of 433 I.U./mg of protein, i.e. a 188-fold purification with an overall yield of 33%. The homogeneity of this preparation was verified by sodium dodecyl sulphate polyacrylamide gel electrophoresis and double immunodiffusion in Ouchterlony plates. Anti-M2-PK antibodies obtained from rabbit neutralized the enzyme activity. Their specificity with regard to other types of PK showed that anti-M2-PK also reacted with M1-PK but not with R-PK.     

Selective determination of trimethylamine in air by liquid chromatography using solid phase extraction cartridges for sampling

The selective determination of trimethylamine (TMA) in air by liquid chromatography is reported. Sampling is effected by flushing air through C18-packed solid-phase extraction (SPE) cartridges at a flow rate of 15 mL/min for 15 min. Next, TMA is desorbed from the cartridges and injected into the chromatographic system. The analyte is then selectively retained on a precolumn (20 mm x 2.1 mm i.d., packed with 30 microm, Hypersil C18 phase), and derivatized on-line by injecting 9-fluorenylmethyl chloroformate (FMOC). Finally, the TMA-FMOC derivative is transferred to the analytical column (125 mm x 4 mm i.d., LiChrospher 100 RP18, 5 microm), and monitored at 262 nm. The method was applied to the measurement of TMA in air in the 0.25-2.5 microg interval (equivalent to concentrations of TMA of 1.1-11 mg/m3), providing good linearity, reproducibility and accuracy. The mean recovery of TMA was (96 +/- 7%) (n = 12), and the limit of detection was 0.05 microg. The proposed procedure allows the selective determination of TMA in the presence of other primary and secondary short-chain aliphatic amines.     

Applicability of the H NMR chemical shifts computed by the ab initio/GIAO-HF methodology to the study of geometrical features of Zn-porphyrin dimers

Several porphyrin dimers have been newly designed and synthesized to construct assemblies with 1,4-diazabicyclo[2.2.2]octane (DABCO) as a bidentate binding ligand. Semi empirical (AM1) and ab initio calculations have been used to study the assemblies generated by the organization of dimers and DABCO, including the computation of ¹H NMR complexation-induced chemical shifts using the ab initio/GIAO methodology. The diagnostic capacity of the theoretical method has been applied to explain experimental results and geometrical features of the complexes.     

Water concentration profiles in membranes measured by ESEEM of spin-labeled lipids

Electron spin-echo envelope modulation (ESEEM) spectroscopy of phospholipids spin-labeled systematically down the sn-2 chain was used to detect the penetration of water (D2O) into bilayer membranes of dipalmitoyl phosphatidylcholine with and without 50 mol % cholesterol. Three-pulse stimulated echoes allow the resolution of two superimposed 2H-ESEEM spectral components of different widths, for spin labels located in the upper part of the lipid chains. Quantum chemical calculations (DFT) and ESEEM simulations assign the broad spectral component to one or two D2O molecules that are directly hydrogen bonded to the N-O group of the spin label. Classical ESEEM simulations establish that the narrow spectral component arises from nonbonded water (D2O) molecules that are free in the hydrocarbon chain region of the bilayer membrane. The amplitudes of the broad 2H-ESEEM spectral component correlate directly with those of the narrow component for spin labels at different positions down the lipid chain, reflecting the local H-bonding equilibria. The D2O-ESEEM amplitudes decrease with position down the chain toward the bilayer center, displaying a sigmoidal dependence on position that is characteristic of transmembrane polarity profiles established by other less direct spin-labeling methods. The midpoint of the sigmoidal profile is shifted toward the membrane center for membranes without cholesterol, relative to those with cholesterol, and the D2O-ESEEM amplitude in the outer regions of the chain is greater in the presence of cholesterol than in its absence. For both membrane types, the D2O amplitude is almost vanishingly small at the bilayer center. The water-penetration profiles reverse correlate with the lipid-chain packing density, as reflected by 1H-ESEEM intensities from protons of the membrane matrix. An analysis of the H-bonding equilibria provides essential information on the binding of water molecules to H-bond acceptors within the hydrophobic interior of membranes. For membranes containing cholesterol, approximately 40% of the nitroxides in the region adjacent to the lipid headgroups are H bonded to water, of which ca. 15% are doubly H bonded. Corresponding H-bonded populations in membranes without cholesterol are ca. 20%, of which ca. 6% are doubly bonded.     

Fused Mesoionic Heterocycles: Synthesis of 1,2,4-triazolo(4,3-b)-1,2,4-triazole derivatives

A number of mesoionic compounds derivatives of the bicyclic system 1,2,4-triazolo(4,3-b)-1,2,4-triazole have been prepared from 4-amino-1-methyl-3,5-bis(methylthio)-1,2,4-triazolium iodide and aryl isothiocyanates.     

Water adsorption on and desorption from crystalline copolymers of vinylidene fluoride with trifluoroethylene

Water adsorption and absorption on crystalline polyvinylidene fluoride with 30% trifluoroethylene, P(VDF-TrFE, 70:30), was examined by thermal desorption spectroscopy. Two distinctly different water adsorption sites are identified: one adsorbed species that resembles ice and another species that interacts more strongly with the polymer thin film. The existence of the latter species is consistent with X-ray diffraction studies of water absorbed into the bulk of copolymers of polyvinylidene fluoride with trifluoroethylene crystalline thin films. There are strong steric effects observed in the angle-resolved thermal desorption that may be a result of the large polymer thin film surface dipoles.