Automated two-dimensional separation flow system with electrochemical preconcentration, stripping, capillary electrophoresis and contactless conductivity detection for trace metal ion analysis

This paper describes the automation of a fully electrochemical system for preconcentration, cleanup, separation and detection, comprising the hyphenation of a thin layer electrochemical flow cell with CE coupled with contactless conductivity detection (CE-C(4)D). Traces of heavy metal ions were extracted from the pulsed-flowing sample and accumulated on a glassy carbon working electrode by electroreduction for some minutes. Anodic stripping of the accumulated metals was synchronized with hydrodynamic injection into the capillary. The effect of the angle of the slant polished tip of the CE capillary and its orientation against the working electrode in the electrochemical preconcentration (EPC) flow cell and of the accumulation time were studied, aiming at maximum CE-C(4)D signal enhancement. After 6 min of EPC, enhancement factors close to 50 times were obtained for thallium, lead, cadmium and copper ions, and about 16 for zinc ions. Limits of detection below 25 nmol/L were estimated for all target analytes but zinc. A second separation dimension was added to the CE separation capabilities by staircase scanning of the potentiostatic deposition and/or stripping potentials of metal ions, as implemented with the EPC-CE-C(4)D flow system. A matrix exchange between the deposition and stripping steps, highly valuable for sample cleanup, can be straightforwardly programmed with the multi-pumping flow management system. The automated simultaneous determination of the traces of five accumulable heavy metals together with four non-accumulated alkaline and alkaline earth metals in a single run was demonstrated, to highlight the potentiality of the system.

Systematic structural studies of iron superoxide dismutases from human parasites and a statistical coupling analysis of metal binding specificity

Superoxide dismutases (SODs) are a crucial class of enzymes in the combat against intracellular free radical damage. They eliminate superoxide radicals by converting them into hydrogen peroxide and oxygen. In spite of their very different life cycles and infection strategies, the human parasites Plasmodium falciparum, Trypanosoma cruzi and Trypanosoma brucei are known to be sensitive to oxidative stress. Thus the parasite Fe-SODs have become attractive targets for novel drug development. Here we report the crystal structures of FeSODs from the trypanosomes T. brucei at 2.0 angstrom and T. cruzi at 1.9 angstrom resolution, and that from P. falciparum at a higher resolution (2.0 angstrom) to that previously reported. The homodimeric enzymes are compared to the related human MnSOD with particular attention to structural aspects which are relevant for drug design. Although the structures possess a very similar overall fold, differences between the enzymes at the entrance to the channel which leads to the active site could be identified. These lead to a slightly broader and more positively charged cavity in the parasite enzymes. Furthermore, a statistical coupling analysis (SCA) for the whole Fe/MnSOD family reveals different patterns of residue coupling for Mn and Fe SODs, as well as for the dimeric and tetrameric states. In both cases, the statistically coupled residues lie adjacent to the conserved core surrounding the metal center and may be expected to be responsible for its fine tuning, leading to metal ion specificity.

Langmuir and Langmuir-Blodgett (LB) films of tetrapyridyl metalloporphyrins

We report on the formation of Langmuir films of 5,10,15,20-tetra(4-pyridyl) 21H,23H-porphine,hereafter named tetrapyridyl porphyrins with distinct central ions (2H(+), Zn(2+), Cu(2+), Ni(2+)). The films were characterized with surface pressure and surface potential isotherms and in situ UV-vis absorbance. The measurements indicated strong aggregation of porphyrin monomers at the air-water interface, with a red shift of the Soret band in comparison with the spectrum obtained from CHCl(3) solutions. The shift was larger for the non-substituted H(2)TPyP, and depended on the metal ion. Significantly, aggregation occurred right after spreading of the Langmuir film, with on further shifts in the UV-vis spectra upon compression of the film, or even after transferring them onto solid substrates in the form of Langmuir-Blodgett (LB) films. The buildup of LB films from H(2)TPyP and ZnTPyP was monitored with UV-vis spectroscopy, indicating an equal amount of material deposited in each deposition step. Using FTIR in the transmission and reflection modes, we inferred that the H(2)TPyP molecules exhibit no preferential orientation in the LB films, while for ZnTPyP there is preferential orientation, with the porphyrin molecules anchored to the substrate by the lateral pyridyl groups. (C) 2008 Elsevier B.V. All rights reserved.

Combined Crystallographic and Solution Molecular Dynamics Study of Allosteric Effects in Ester and Ketone p-tert-Butylcalix[4]arene Derivatives and Their Complexes with Acetonitrile, Cd(II), and Pb(II)

We describe here a procedure to bridge the gap in the field of calixarene physicochemistry between solid-state atomic-resolution structural information and the liquid-state low-resolution thermodynamics and spectroscopic data. We use MD simulations to study the kinetics and energetics involved in the complexation of lower rim calix[4]arene derivatives (L), containing bidentate ester (1) and ketone (2) pendant groups, with acetonitrile molecule (MeCN) and Cd2+ and Pb2+ ions (M2+) in acetonitrile solution. On one hand, we found that the prior inclusion of MeCN into the calix to form a L(MeCN) adduct has only a weak effect in preorganizing the hydrophilic cavity toward metal ion binding. On the other hand, the strong ion-hydrophilic cavity interaction produces a wide open calix which enhances the binding of one MeCN molecule (allosteric effect) to stabilize the whole (M2+)1(MeCN) bifunctional complex. We reach two major conclusions: (i) the MD results for the (M2+)1(MeCN) binding are in close agreement with the ""endo"", fully encapsulated, metal complex found by X-ray diffraction and in vacuo MD calculations, and (ii) the MD structure for the more flexible 2 ligand, however, differs from the also endo solid-state molecule. In fact, it shows strong solvation effects at the calixarene lower bore by competing MeCN molecules that share the metal coordination sphere with the four C=O oxygens of an ""exo"" (M2+)2(MeCN) complex.

Study of the phenanthroline-Mn-imidazole bonding in Mn(I) triscarbonyl complex: A X-ray and DFT computational analysis

355 nm light irradiation of fac-[Mn(CO)(3)(phen)(imH)](+) (fac-1) produces the mer-1 isomer and a long lived radical which can be efficiently trapped by electron acceptor molecules. EPR experiments shows that when excited, the manganese(I) complex can be readily oxidized by one-electron process to produce Mn(II) and phen(.-). In the present study, DFT calculations have been used to investigated the photochemical isomerization of the parent Mn(I) complex and to characterize the electronic structures of the long lived radical. The theoretical calculations have been performed on both the fac-1 and mer-1 species as well as on their one electron oxidized species fac-1+ and mer-1+ for the lowest spin configurations (S = 1/2) and fac-6 and mer-6 (S = 5/2) for the highest one to characterize these complexes. In particular, we used a charge decomposition analysis (CDA) and a natural bonding orbital (NBO) to have a better understanding of the chemical bonding in terms of the nature of electronic interactions. The observed variations in geometry and bond energies with an increasing oxidation state in the central metal ion are interpreted in terms of changes in the nature of metal-ligand bonding interactions. The X-ray structure of fac-1 is also described. (C) 2011 Elsevier B.V. All rights reserved.

The stability and aggregation properties of the GTPase domain from human SEPT4

The septins are a family of conserved proteins involved in cytokinesis and cortical organization. An increasing amount of data implicates different septins in diverse pathological conditions including neurodegenerative disorders, neoplasia and infections. Human SEPT4 is a member of this family and its tissue-specific ectopic expression profile in colorectal and urologic cancer makes it a useful diagnostic biomarker. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediate which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, we examined the effects of protein concentration, pH and metals ions on the aggregation process of recombinant SEPT4-G using a series of biophysical techniques, which were also employed to study chemical unfolding and stability. Divalent metal ions caused significant acceleration to the rate of SEPT4-G aggregation. Urea induced unfolding was shown to proceed via the formation of a partially unfolded intermediate state which unfolds further at higher urea concentrations. The intermediate is a compact dimer which is unable to bind GTR At 1 M urea concentration, the intermediate state was plagued by irreversible aggregation at temperatures above 30 degrees C. However, higher urea concentration resulted in a marked decay of the aggregation, indicating that the partially folded structures may be necessary for the formation of these aggregates. The results presented here are consistent with the recently determined crystal structure of human septins and shed light on the aggregation properties of SEPT4 pertinent to its involvement in neurodegenerative disease. (C) 2008 Elsevier B.V. All rights reserved.

Ca(2+)-mediated thiolysis of peptide-resin linkage as an option for preparing protected peptide thioesters

A mild new procedure for preparing protected peptide thioesters, based oil Ca(2+)-assisted thiolysis of peptide-Kaiser oxime resin (KOR) linkage, is described. Ac-Ile-Ser(Bzl)-Asp(OcHx)-SR (Ac: acetyl; Bzl: benzyl; cHx: cyclohexyl), model peptide, was readily released from the resin by incubating the peptide-KOR at 60 degrees C in mixtures of DMF with n-butanethiol [R = (CH(2))(3)CH(3)] or ethyl 3-mercaptopropionate [R = (CH(2))(2)COOCHCH(3)] containing Ca(CH(3)COO)(2). After serine and aspartic acid side-chain deprotection under acid conditions, Ac-Ile-Ser-Asp-S(CH(2))(2)COOCH(2)CH(3) was successfully obtained with good quality and high yield. This type of C-terminal modified peptide may act as an excellent acyl donor in peptide segment condensation by the thioester method, native chemical ligation and enzymatic methods. (c) 2008 Elsevier Ltd. All rights reserved.

Oxindole-Schiff base copper(II) complexes interactions with human serum albumin: Spectroscopic, oxidative damage, and computational studies

CD and EPR were used to characterize interactions of oxindole-Schiff base copper(II) complexes with human serum albumin (HSA). These imine ligands form very stable complexes with copper, and can efficiently compete for this metal ion towards the specific N-terminal binding site of the protein, consisting of the amino acid sequence Asp-Ala-His. Relative stability constants for the corresponding complexes were estimated from CD data, using the protein as competitive ligand, with values of log K(CuL) in the range 15.7-18.1, very close to that of [Cu(HSA)] itself, with log K(CuHSA) 16.2. Some of the complexes are also able to interfere in the a-helix structure of the protein, while others seem not to affect it. EPR spectra corroborate those results, indicating at least two different metal species in solution, depending on the imine ligand. Oxidative damage to the protein after incubation with these copper(II) complexes, particularly in the presence of hydrogen peroxide, was monitored by carbonyl groups formation, and was observed to be more severe when conformational features of the protein were modified. Complementary EPR spin-trapping data indicated significant formation of hydroxyl and carbon centered radicals, consistent with an oxidative mechanism. Theoretical calculations at density functional theory (DFT) level were employed to evaluate Cu(II)-L binding energies, L -> Cu(II) donation, and Cu(II) -> L back-donation, by considering the Schiff bases and the N-terminal site of HSA as ligands. These results complement previous studies on cytotoxicity, nuclease and pro-apoptotic properties of this kind of copper(II) complexes, providing additional information about their possibilities of transport and disposition in blood plasma. (C) 2009 Elsevier Inc. All rights reserved.

Photoluminescence behavior of Eu(3+) ion doped into gamma- and alpha-alumina systems prepared by combustion, ceramic and Pechini methods

Al(2)O(3):Eu(3+)(1%) samples were prepared by combustion, ceramic, and Pechini methods annealed from 400 to 1400 degrees C. XRD patterns indicate that samples heated up to 1000 degrees C present disordered character of activated alumina (gamma-Al(2)O(3)). However, alpha-Al(2)O(3) phase showed high crystallinity and thermostability at 1200-1400 degrees C. The sample characterizations were also carried out by means of infrared spectroscopy (IR), scanning electron microscopy (SEM) and specific surface areas analysis (BET method). Excitation spectra of Al(2)O(3):Eu(3+) samples present broaden bands attributed to defects of Al(2)O(3) matrices and to LMCT state of O -> Eu(3+), however, the narrow bands are assigned to (7)F(0) -> (5)D(J),(5)H(J) and (5)L(J) transitions of Eu(3+) ion. Emission spectra of samples calcined up to 1000 degrees C show broaden bands for (5)D(0) -> (7)F(J) transitions of Eu(3+) ion suggesting that the rare earth ion is in different symmetry sites showed by inhomogeneous line broadening of bands, confirming the predominance of the gamma-alumina phase. For all samples heated from 1200 to 1400 degrees C the spectra exhibit narrow (5)D(0) -> (7)F(J) transitions of Eu(3+) ion indicating the conversion of gamma to alpha-Al(2)O(3) phases, a high intensity narrow peak around 695 nm assigned to R lines of Cr(3+) ion is shown. Al(2)O(3):Eu(3+) heated up to 1100 degrees C presents an increase in the Omega(2) intensity parameter with the increase of temperatures enhancing the covalent character of metal-donor interaction. The disordered structural systems present the highest values of emission quantum efficiencies (eta). CIE coordinates of Al(2)O(3):Eu(3+) are also discussed. (C) 2007 Elsevier Inc. All rights reserved.

X-ray photoelectron spectroscopy analysis of zirconium nitride-like films prepared on Si(100) substrates by ion beam assisted deposition

Thin zirconium nitride films were prepared on Si(l 00) substrates at room temperature by ion beam assisted deposition with a 2 keV nitrogen ion beam. Arrival rate ratios ARR(N/Zr) used were 0.19, 0.39, 0.92, and 1.86. The chemical composition and bonding structure of the films were analyzed with X-ray photoelectron spectroscopy (XPS). Deconvolution results for Zr 3d, Zr 3p(3/2), N 1s, O 1s, and C 1s XPS spectra indicated self-consistently the presence of metal Zr-0, nitride ZrN, oxide ZrO2, oxymnide Zr2N2O, and carbide ZrC phases, and the amounts of these compounds were influenced by ARR(N/Zr). The chemical composition ratio N/Zr in the film increased with increasing ARR(N/Zr) until ARR(N/Zr) reached 0.92, reflecting the high reactivity of nitrogen in the ion beam, and stayed almost constant for ARR(N/Zr) >= 1, the excess nitrogen being rejected from the growing film. A considerable incorporation of contaminant oxygen and carbon into the depositing film was attributed to the getter effect of zirconium. (C) 2007 Elsevier B.V. All rights reserved.

Ion beam assisted deposition of an organic light emitting diode electrode

This work presents the electro-optical characterization of metal-organic interfaces prepared by the Ion Beam Assisted Deposition (IBAD) method. IBAD applied in this work combines simultaneously metallic film deposition and bombardment with an independently controlled ion beam, allowing different penetration of the ions and the evaporated metallic elements into the polymer. The result is a hybrid, non-abrupt interface, where polymer, metal and ion coexists. We used an organic light emitting diode, which has a typical vertical-architecture, for the interface characterization: Glass/Indium Tin Oxide (ITO)/Poly[ethylene-dioxythiophene/poly{styrenesulfonicacid}]) (PEDOT:PSS) /Emitting Polymer/Metal. The emitting polymer layer comprised of the Poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFO) and the metal layer of aluminum prepared with different Ar(+) ion energies varying in the range from 0 to 1000 eV. Photoluminescence, Current-Voltage and Electroluminescence measurements were used to study the emission and electron injection properties. Changes of these properties were related with the damage caused by the energetic ions and the metal penetration into the polymer. Computer simulations of hybrid interface damage and metal penetration were confronted with experimental data. (C) 2010 Elsevier B.V. All rights reserved.

Efficiency in the loading of a sodium magneto-optical trap from alkali metal dispensers

We study the loading of sodium atoms into a magneto-optical trap from current-controlled sodium metal dispensers. Contrary to what was previously reported [V. Wippel, C. Binder, W. Huber, L Windholz, M. Allegrini, F. Fuso, E. Arimondo, Eur. Phys. J. D 17 (2001) 2851 we demonstrate a significantly higher number of trapped atoms that make Na dispensers a feasible source of atoms for cold-atom studies. The inherent rise in pressure that naturally arises from metal dispensers as they are heated to release atoms is partially controlled by placing the metal dispensers near the pumping port where an ion pump is connected. We also study the effects of placing the sodium dispensers at different distances from the main vacuum chamber where the atoms are trapped and the effectiveness of using a Zeeman slower to cool the atoms as they emerge from the dispensers. We observe trapping of up to 1.9 x 10(8) atoms, which is significantly higher by almost three orders of magnitude than previously reported experiments. (C) 2008 Elsevier B.V. All rights reserved.

1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation

The alpha-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB`s cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37 degrees C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other alpha-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml(-1)) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(center dot), and those with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(center dot) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 mu M) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity. (C) 2011 Elsevier Inc. All rights reserved.

PREDICTION OF METAL CATION TOXICITY TO THE BIOLUMINESCENT FUNGUS GERRONEMA VIRIDILUCENS

A correlation between the physicochemical properties of mono- [Li(I), K(I), Na(I)] and divalent [Cd(II), Cu(II), Mn(II), Ni(II), Co(II), Zn(II), Mg(II), Ca(II)] metal cations and their toxicity (evaluated by the free ion median effective concentration. EC50(F)) to the naturally bioluminescent fungus Gerronema viridilucens has been studied using the quantitative ion character activity relationship (QICAR) approach. Among the 11 ionic parameters used in the current study, a univariate model based on the covalent index (X(m)(2)r) proved to be the most adequate for prediction of fungal metal toxicity evaluated by the logarithm of free ion median effective concentration (log EC50(F)): log EC50(F) = 4.243 (+/-0.243) -1.268 (+/-0.125).X(m)(2)r (adj-R(2) = 0.9113, Alkaike information criterion [AIC] = 60.42). Additional two- and three-variable models were also tested and proved less suitable to fit the experimental data. These results indicate that covalent bonding is a good indicator of metal inherent toxicity to bioluminescent fungi. Furthermore, the toxicity of additional metal ions [Ag(I), Cs(I), Sr(II), Ba(II), Fe(II), Hg(II), and Pb(II)] to G. viridilucens was predicted, and Pb was found to be the most toxic metal to this bioluminescent fungus (EC50(F)): Pb(II) > Ag(I) > Hg(I) > Cd(II) > Cu(II) > Co(II) Ni(II) > Mn(II) > Fe(II) approximate to Zn(II) > Mg(II) approximate to Ba(II) approximate to Cs(I) > Li(I) > K(I) approximate to Na(I) approximate to Sr(II)> Ca(II). Environ. Toxicol. Chem. 2010;29:2177-2181. (C) 2010 SETAC