Electrically induced fluorescence Fe3+ sensing behavior of nanostructured Tiron doped polypyrrole.

Nanostructured polypyrrole (PPy) film doped with Tiron was electrodeposited from aqueous solution on the surface of transparent electrode and used for sensitive, selective and rapid electrically controlled fluorescence detection of Fe3+ in aqueous media. The fluorescence intensity of PPy-Tiron film decreases linearly in the presence of Fe3+ by applying negative potential over a concentration range from 5.0 × 10-8 to 1.0 × 10-6 mol L-1, with a relatively fast response time of less than 30 s at pH 7.4. The detection is not affected by the coexistence of other competitive metal ions such as Al3+, Ce3+, Tl3+, La3+, Bi3+, Cr2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+, Na+, K+, Mg2+, Ca2+, Sr2+ and Ba2+. The proposed electro-fluorescence sensor has a potential application to the determination of Fe3+ in environmental and biological systems. The fluorescent thin film sensor was also used as a novel probe for Fe3+/Fe2+ speciation in aqueous solution.

Experimental and Computational Evidence for the Reduction Mechanisms of Aromatic N-oxides by Aqueous Fe(II)-Tiron Complex.

A combined experimental-theoretical approach was taken to elucidate the reduction mechanisms of five representative aromatic N-oxides (ANOs) by Fe(II)-tiron complex and to identify the rate-limiting step. Based on the possible types of complexes formed with the reductant, three groups of ANOs were studied: type I refers to those forming 5-membered ring complexes through the N and O atoms on the side chain; type II refers to those forming 6-membered ring complexes through the N-oxide O atom and the O atom on the side chain; and type III refers to complexation through the N-oxide O atom only. Density functional theory calculations suggested that the elementary reactions, including protonation, N-O bond cleavage, and the second electron transfer processes, are barrierless, indicating that the first electron transfer is rate-limiting. Consistent with the theoretical results, the experimental solvent isotope effect, KIEH, for the reduction of quinoline N-oxide (a type III ANO) was obtained to be 1.072 ± 0.025, suggesting protonation was not involved in the rate-limiting step. The measured nitrogen kinetic isotope effect, KIEN, for the reduction of pyridine N-oxide (a type III ANO) (1.022 ± 0.006) is in good agreement with the calculated KIEN for its first electron transfer (1.011-1.028), confirming that the first electron transfer is rate-limiting. Electrochemical cell experiments demonstrated that the electron transfer process can be facilitated significantly by type I complexation with FeL2(6-) (1:2 Fe(II)-tiron complex), to some extent by type II complexation with free Fe(II), but not by weak type III complexation.

Oxidative DNA cleavage by Cu(II) complexes: Effect of periphery substituent groups.

A series of structurally-related [Cu(R-benzyl-dipicolylamine)(NO3)2] complexes, where R=methoxy- (1), methyl- (2), H- (3), fluoro- (4), and nitro-group (5), were synthesized, and their activity on DNA cleavage was investigated by linear dichroism (LD) and electrophoresis. The addition of a benzyl group to the dipicolylamine ligand of the [Cu(dipicolylamine)(NO3)2] complex (A), i.e., the [Cu(benzyl-dipicolylamine)(NO3)2] complex (3), caused significant enhancement in the efficiency of oxidative cleavage of both super-coiled (sc) and double stranded (ds) DNA, as evidenced by the electrophoresis pattern and faster decrease in the LD intensity at 260nm. The efficiency in DNA cleavage was also altered with further modifications of the benzyl group by the introduction of various substituents at the para-position. The cleavage efficiency appeared to be the largest when the methyl group was attached. The order of efficiency in DNA cleavage was methyl>methoxy≈H>fluoro≈nitro group. When an electron-withdrawing group was introduced, the cleavage efficiency decreased remarkably. The reactive oxygen species involved in the cleavage process were the superoxide radical and singlet oxygen. A possible mechanism for this variation in the DNA cleavage efficiency was proposed.

Modification of glassy carbon electrode with a bilayer of multiwalled carbon nanotube/tiron-doped polypyrrole: Application to sensitive voltammetric determination of acyclovir.

A novel voltammetric sensor based on glassy carbon electrode (GCE) modified with a thin film of multi-walled carbon nanotubes (MWCNTs) coated with an electropolymerized layer of tiron-doped polypyrrole was developed and the resulting electrode was applied for the determination of acyclovir (ACV). The surface morphology and property of the modified electrode were characterized by field emission scanning electron microscopy and electrochemical impedance spectroscopy techniques. The electrochemical performance of the modified electrode was investigated by means of linear sweep voltammetry (LSV). The effect of several experimental variables, such as pH of the supporting electrolyte, drop size of the cast MWCNTssuspension, number of electropolymerization cycles and accumulation time was optimized by monitoring the LSV response of the modified electrode toward ACV. The best response was observed at pH7.0 after accumulation at open circuit for 160 s. Under the optimized conditions, a significant electrochemical improvement was observed toward the electrooxidation of ACV on the modified electrode surface relative to the bare GCE, resulting in a wide linear dynamic range (0.03-10.0µ M) and a low detection limit (10.0 nM) for ACV. Besides high sensitivity, the sensor represented high stability and good reproducibility for ACV analysis, and provided satisfactory results for the determination of this compound in pharmaceutical and clinical preparations.

A highly sensitive fluorescence probe for metallothioneins based on tiron-copper complex.

The fabrication of tiron-copper complex as a novel fluorescence probe for the sensitive directly detection of metallothioneins at nanomolar levels was demonstrated. In Britton-Robinson (B-R) buffer (pH 7.50), the interaction of bis(tiron)copper(II) complex cation [Cu(tiron)2](2+) and metallothioneins enhanced the fluorescence intensity of the system. The fluorescence enhancement at 347 nm was proportional to the concentration of metallothioneins. The mechanism was studied and discussed in terms of the fluorescence spectra. Under the optimal experimental conditions, at 347 nm, there was a linear relationship between the fluorescence intensity and the concentration of the metallothioneins in the range of 8.80 × 10(-9)-7.70 × 10(-7)mol L(-1), with a correlation coefficient of r=0.995 and detection limit 2.60 × 10(-9)mol L(-1). The relative standard deviation was 0.77% (n=11), and the average recovery 94.4%. The method proposed was successfully reliable, selective and sensitive in determining of trace metallothioneins in fish visceral organ samples with the results in good agreement with those obtained by HPLC.

Complexation facilitated reduction of aromatic N-oxides by aqueous Fe(II)-tiron complex: reaction kinetics and mechanisms.

Rapid reduction of carbadox (CDX), olaquindox and several other aromatic N-oxides were investigated in aqueous solution containing Fe(II) and tiron. Consistent with previous work, the 1:2 Fe(II)-tiron complex, FeL2(6-), is the dominant reactive species as its concentration linearly correlates with the observed rate constant kobs under various conditions. The N-oxides without any side chains were much less reactive, suggesting direct reduction of the N-oxides is slow. UV-vis spectra suggest FeL2(6-) likely forms 5- or 7-membered rings with CDX and olaquindox through the N and O atoms on the side chain. The formed inner-sphere complexes significantly facilitated electron transfer from FeL2(6-) to the N-oxides. Reduction products of the N-oxides were identified by HPLC/QToF-MS to be the deoxygenated analogs. QSAR analysis indicated neither the first electron transfer nor N-O bond cleavage is the rate-limiting step. Calculations of the atomic spin densities of the anionic N-oxides confirmed the extensive delocalization between the aromatic ring and the side chain, suggesting complex formation can significantly affect the reduction kinetics. Our results suggest the complexation facilitated N-oxide reduction by Fe(II)-tiron involves a free radical mechanism, and the subsequent deoxygenation might also benefit from the weak complexation of Fe(II) with the N-oxide O atom.

A flow system with zone merging and zone trapping in the main reactor applied to spectrophotometric catalytic determination of cobalt in grasses.

A flow system with zone merging and zone trapping in the main reactor was proposed. The sample and reagent inserted aliquots merge together and the resulting zone is directed towards a displaceable reactor inside which its most concentrated portion is trapped. After the pre-set TRAP period, the handled sample is released towards detection. A comparison with an analogous flow system exploiting zone stopping revealed the superior characteristics of sampling rate and system operation; moreover, the sample inserted volume plays little influence on sampling rate. The system was applied to the spectrophotometric determination of cobalt in pastures, and enhanced figures of merit (sampling rate=18 h(-1); peak height r.s.d.=0.7%, detection limit=0.046 µg L(-1) Co; reagent consumption=330 µg of Tiron per measurement; 98%

Enhanced mixing and plume containment in porous media under time-dependent oscillatory flow.

Solute transport experiments were conducted in a decimeter scale flow cell packed with sand to study the potential for enhanced mixing of solutes in porous media and improved containment of injected plumes under multiple pumping-well driven, time-dependent oscillatory flow with respect to constant flow. Real-time imaging of the colorimetric reaction of Tiron (1,2-dihydroxybenzene-3,5-disulfonic acid) and molybdate was used to quantify mixing, whereas fluorescein was used to better examine plume size. Results from the small scale experiments clearly demonstrated the enhanced mixing of solutes under low Reynolds number oscillatory flow (a factor of 2 with respect to constant flow in homogeneous sand and a factor of 3 in layered sand), as the result of increased contact interface for solute diffusion. Further, the injected solute plume was better contained under oscillatory flow (25% less area with respect to constant flow in homogeneous sand) due to the cancellation of advective transport at each well over time. Enhanced mixing under oscillatory flow may enhance the processes of chemical and biological remediation. Furthermore, improved plume containment under oscillatory flow may require smaller amounts of chemicals to be injected during aquifer remediation.

Genetically engineering transferrin to improve its in vitro ability to deliver cytotoxins.

We previously demonstrated that decreasing the iron release rate of transferrin (Tf), by replacing the synergistic anion carbonate with oxalate, increases its in vitro drug carrier efficacy in HeLa cells. In the current work, the utility of this strategy has been further explored by generating two Tf mutants, K206E/R632A Tf and K206E/K534A Tf, exhibiting different degrees of iron release inhibition. The intracellular trafficking behavior of these Tf mutants has been assessed by measuring their association with HeLa cells. Compared to native Tf, the cellular association of K206E/R632A Tf and K206E/K534A Tf increased by 126 and 250%, respectively. Surface plasmon resonance studies clearly indicate that this increase in cellular association is due to a decrease in the iron release rate and not to differences in binding affinity of the mutants to the Tf receptor (TfR). Diphtheria toxin (DT) conjugates of K206E/R632A Tf and K206E/K534A Tf showed significantly increased cytotoxicity against HeLa cells with IC(50) values of 1.00 pM and 0.93 pM, respectively, compared to a value of 1.73 pM for the native Tf conjugate. Besides further validating our strategy of inhibiting iron release, these Tf mutants provide proof-of-principle that site-directed mutagenesis offers an alternative method for improving the drug carrier efficacy of Tf.

Use of proliferation tests to evaluate the effects of complexing agents on beryllium toxicity.

Occupational exposure to beryllium may cause chronic beryllium disease (CBD), a granulomatous interstitial pneumonitis caused by a cell-mediated immune response with delayed hypersensitivity initiated by an electrostatic interaction with the MHC class II human leukocyte antigen (HLA). Increased research efforts focus on the development of a CBD treatment by chelation therapy. This work presents an in vitro evaluation of the beneficial effects of beryllium chelation with different organic substrates. We have used a standard beryllium lymphocyte proliferation test (BeLPT) adapted for mouse splenocytes. Three complexing agents, 4,5-dihydroxy-1,3-benzenedisulfonic acid (tiron), nitrilotripropionic acid (NTP) and nitrilotriacetic acid (NTA), were tested using different protocols of the splenocyte proliferation test (SPT). We studied their corrective effect (beryllium pre-exposed splenocytes), their protective effect (ligand pre-exposed splenocytes) and their combined effects at fixed Be:L ratio of 1:2, at fixed Be concentration and at fixed L concentration. We also studied the effect of tiron in preventing splenocyte sensitization to beryllium. All three complexing agents showed a corrective effect and proved efficient in the combined effects, except NTA in the fixed Be:L ratio. Only NTP and tiron showed a significant protection at lower beryllium concentrations, while NTA was not significant. Splenocytes pre-exposed to chelated beryllium did not show sensitization while splenocytes pre-exposed to beryllium were sensitized. We observed a strong correlation between the efficiency of the complexing agent and its affinity towards beryllium. Both tiron and NTP showed a similar affinity towards the beryllium ion that is 10(7) higher than that of NTA.

Nicotinamide adenine dinucleotide phosphate (reduced form) oxidase is important for LPS-induced endothelial cell activation.

Activation of the endothelium plays an important role in the innate immune response. This process is associated with an increase in the production of superoxide (O2-) by nicotinamide adenine dinucleotide phosphate (reduced form; NADPH) oxidase. Our objective was to determine if O2- from NADPH oxidase contributes to activation of human umbilical vein endothelial cells by LPS as it does for TNF-alpha. We used the adhesion molecule intracellular adhesion molecule 1 and cytokine IL-8 as indicators of human umbilical vein endothelial cell activation and measured O2- production with chemiluminescence. LPS increased baseline and NADPH-stimulated O2- production. The increase was reduced by tiron, a protein kinase C inhibitor (bisindolylmaleimide I hydrochloride), the flavin inhibitor (diphenylene iodonium), and by a short interfering RNA against the p22phox component of NADPH oxidase. Inhibition of NADPH oxidase with the short interfering RNA reduced the induction by LPS of intracellular adhesion molecule 1 mRNA, protein, and IL-8 release (by enzyme-linked immunosorbent assay). The production of O2- by NADPH oxidase contributes to intracellular signaling by LPS in endothelial cells as it does for TNF-alpha and helps turn on the innate immune response in these cells.

Influence of organic ligands on the reduction of polyhalogenated alkanes by iron(II).

Experimental work demonstrates that polyhalogenated alkanes (PHAs) are rapidly reduced in aqueous solutions containing Fe(II) complexes with organic ligands that possess either catechol or organothiol Lewis base groups in their structure and are representative of extracellular ligands and metal-complexing moieties within humic substances (tiron, 2,3-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid, thioglycolic acid, and 2,3-dimercaptosuccinic acid). In solutions containing Fe(II)-tiron complexes, 1,1,1-trichloroethane (1,1,1-TCA) is reduced quantitativelyto acetaldehyde, a product previously reported for reactions with Cr(II), but not with Fe-based reductants. Observed pseudo-first-order rate constants for 1,1,1-TCA reduction by Fe(II)-organic complexes (k'(obs)) generally increase with increasing pH and ligand concentration when Fe(II) concentration is fixed. For the Fe(II)-tiron system, k'(obs) is linearly correlated with the concentration of the 1:2 Fe(II)-tiron complex (FeL2(6-)), and kinetic trends can be described by k'(obs) = k(FeL2)6- [FeL2(6-)], where k(FeL2)6- is the bimolecular rate constant for PHA reaction with the 1:2 Fe(II)-tiron complex. Comparing reaction rates for 14 polyhalogenated ethanes and methanes reveals linear free energy relationships (LFERs) with molecular descriptors for PHA reduction (D(R-X'), deltaG(0'), and E(LUMO)), with the strongest correlation being obtained using carbon-halogen bond dissociation energies, D(R-X'). The collective experimental results are consistent with a dissociative one-electron transfer process occurring during the rate-limiting step.

Calmagite dye oxidation using in situ generated hydrogen peroxide catalysed by manganese(II) ions.

Hydrogen peroxide (H(2)O(2)) generated from the manganese(II) catalysed reduction of dioxygen has been shown to efficiently oxidize Calmagite (3-hydroxy-4-(2-hydroxy-5-methylphenylazo)naphthalene-1-sulfonic acid) in aqueous solution at pH 8.0 and 20 +/- 1 degrees C with de-protonated Tiron (1,2-dihydroxybenzene-3,5-disulfonate, disodium salt) acting as an essential co-ligand.

Determination of alkali-labile phosphoprotein phosphorus from fish plasma using the Tb(3+)-tiron complex as a fluorescence probe.

A sensitive method based on the fluorescence quenching effect of the Tb(3+)-Tiron complex is proposed for the determination of alkali-labile phosphoprotein phosphorus (ALP) released from fish plasma. The detection limit was 5.4 ng/ml (S/N = 2), and the relative standard deviation of the quenching effect (6 replicates) was 4.6%. The results obtained by the proposed method were in good agreement with those obtained by the colorimetric assay. The advantages of the present method are its relatively simple detection procedure, the lack of toxic organic solvents, and high sensitivity.

Iron-sulfur cluster stability. Kinetics and mechanism of ligand-promoted cluster degradation.

Reactivity studies of iron-sulfur cluster proteins with chelating ligands model the reactivity of cluster scaffold proteins such as ISU, and suggest a rate law [k(obs) = k2[ligand]n/{[ligand]n + K(D)}] consistent with formation of a pre-reaction complex between the Fe-S protein and one chelate ligand.

Abiotic reduction of nitroaromatic compounds by aqueous iron(ll)-catechol complexes.

Complexation of iron(ll) by catechol and thiol ligands leads to the formation of aqueous species that are capable of reducing substituted nitroaromatic compounds (NACs) to the corresponding anilines. No reactions of NACs are observed in FelI-only or ligand-only solutions. In solutions containing FeII and tiron, a model catechol, rates of NAC reduction are heavily dependent on pH, ligand concentration, and ionic strength. Observed pseudo-first-order rate constants (k(obs)) for 4-chloronitrobenzene reduction vary by more than 6 orders of magnitude, and the variability is well described by the expression k(obs) = k(FeL2)(6-) [FeL2(6-)], where [FeL2(6-)] is the concentration of the 1:2 FeII-tiron complex and kFeL2(6-) is the bimolecular rate constant for 4-chloronitrobenzene reaction with this species. The high reactivity of this FeII species is attributed to the low standard one-electron reduction potential of the corresponding FeIII/FeII redox couple (EH0 = -0.509 V vs NHE). The relative reactivity of different NACs can be described by a linear free-energy relationship (LFER) with the one-electron reduction potentials of the NACs, EH1'(ArNO2). The experimentally derived slope of the LFER indicates that electron transfer is rate determining. These findings suggest that FeII-organic complexes may play an important, previously unrecognized, role in the reductive transformation of persistent organic contaminants.

Chemical blockage of the proteasome inhibitory function of bortezomib: impact on tumor cell death.

The proteasome inhibitor bortezomib is emerging as a potent anti-cancer agent. Still, recent clinical trials have revealed a significant secondary toxicity of bortezomib. Consequently, there is much interest in dissecting the mechanism of action of this compound to rationally improve its therapeutic index. The cytotoxic effect of bortezomib is frequently characterized by interfering with downstream events derived from the accumulation of proteasomal targets. Here we identify the first chemical agent able to act upstream of the proteasome to prevent cell killing by bortezomib. Specifically, we show that the polyhydroxyl compound Tiron can function as a competitive inhibitor of bortezomib. This effect of Tiron was surprising, since it is a classical radical spin trap and was expected to scavenge reactive oxygen species produced as a consequence of bortezomib action. The inhibitory effect of Tiron against bortezomib was selective, since it was not shared by other antioxidants, such as vitamin E, MnTBAP, L-N-acetyl-cysteine, and FK-506. Comparative analyses with nonboronated proteasome inhibitors (i.e. MG132) revealed a specificity of Tiron for bortezomib. We exploited this novel feature of Tiron to define the "point of no return" of proteasome inhibition in melanoma cells and to block cell death in a three-dimensional model of human skin. Cells from T-cell lymphoma, breast carcinoma, and non-small cell lung cancer were also responsive to Tiron, suggesting a broad impact of this agent as a bortezomib blocker. These results may have important implications for the analysis of bortezomib in vivo and for the design of drug mixtures containing proteasome inhibitors.

Kinetics of iron release from ferric binding protein (FbpA): mechanistic implications in bacterial periplasm-to-cytosol Fe3+ transport.

The ferric binding protein (FbpA) transports iron across the periplasmic space of certain Gram-negative bacteria and is an important component involved in iron acquisition by pathogenic Neisseria spp. (Neisseria gonorrheae and Neisseria meningitidis). Previous work has demonstrated that the synergistic anion, required for tight Fe(3+) sequestration by FbpA, also plays a key role in inserting Fe(3+) into the FbpA binding site. Here, we investigate the iron release process from various forms of holo-FbpA, Fe(3+)FbpA-X, during the course of a chelator competition reaction using EDTA and Tiron. Fe(3+)FbpA-X represents the protein assembly complex with different synergistic anions, X = PO(4)(3)(-) and NTA. Stepwise mechanisms of Fe(3+) release are proposed on the basis of kinetic profiles of these chelator competition reactions. Fe(3+)FbpA-PO(4) and Fe(3+)FbpA-NTA react differently with EDTA and Tiron during the Fe(3+)-exchange process. EDTA replaces PO(4)(3)(-) and NTA from the first coordination shell of Fe(3+) and acts as a synergistic anion to give a spectroscopically distinguishable intermediate, Fe(3+)FbpA-EDTA, prior to pulling Fe(3+) out of the protein. Tiron, on the other hand, does not act as a synergistic anion but is a more efficient competing chelator as it removes Fe(3+) from FbpA at rate much faster than EDTA. These results reaffirm the contribution of the synergistic anion to the FbpA iron transport process as the anion, in addition to playing a facilitative role in iron binding, appears to have a "gatekeeper" role, thereby modulating the Fe(3+) release process.