Mechanism of the catalysis by Alcaligenes eutrophus H16 hydrogenase of direct electrochemical reduction of NAD+

The mechanism of direct electrochemical reduction of NAD⁺ into NADH catalysed by Alcaligenes eutrophus H16 hydrogenase was analysed in thin layer electrochemical cells with platinum and carbon electrodes. Two phases can be distinguished in the catalytic reaction occurring on platinum electrodes. In the potential range from approximately ?0.620 to ?0.675 V (SCE) direct electron transfer occurred via the diaphorase-like dimer of the hydrogenase. Below ?0.69 V versus SCE the hydrogenase used hydrogen species adsorbed onto the platinum electrode, but no molecular hydrogen was required for this second catalytic phase. The mechanism was quite similar to those which had been previously determined for Rhodococcus opacus hydrogenase. This confirmed the very great similarity of the two enzymes, even if the maximum NAD⁺ reduction rate of 0.36 mM min^?1 obtained here remains lower than those reached with R. opacus hydrogenase in a previous study. Careful analysis of the experimental data obtained on a carbon electrode led to the conclusion that no direct electron transfer was observed on this material under the operating conditions used. On the other hand, the voltammetric experiments performed on a carbon electrode in a thin layer cell showed clearly the occurrence of a catalytic current due to the hydrogenase-catalysed reduction of NAD⁺ by molecular hydrogen. This may be a useful tool for further analysis of the hydrogenase kinetics.     

UV/ozone pretreatment of glassy carbon electrodes

A commercial UV/ozone cleaning device is investigated for use in pretreating glassy-carbon (GC) electrodes. In combination with alumina polishing, UV/ozone pretreatment produces electrodes with high activity for oxidation of catecholamines and ascorbic acid. UV/ozone treatment also reverses the passivating effect of sonicating GC electrodes in protic solvents and minimizes adsorption of dopamine and 4-methyl catechol. UV/ozone treatment also cleans electrodes fouled by electrolysis by-products. Fifteen minutes of UV/ozone treatment produced near complete restoration of activity at a GC electrode passivated by a phenolic polymer film. UV/ozone treatment increases activity by oxidative removal of organic adsorbates and a polishing-produced carbon-powder overlayer. In addition, XPS and cyclic voltammetry indicate that carbon-oxide concentration increases on the surface of the UV/ozone treated carbon electrode as compared to an alumina-polished electrode.     

Direct electron transfer between the heme of cellobiose dehydrogenase and thiol modified gold electrodes

Cellobiose dehydrogenase (CDH) is an extracellular fungal enzyme with two domains, one containing flavin adenine dinucleotide (FAD) and one containing heme. The electrochemistry of CDH, as well as its cleaved FAD- and heme-subunits, was studied using a membrane electrode, i.e. the enzyme was trapped under a permselective membrane on a cystamine or 3-mercaptopropionic acid modified gold electrode. Direct un-mediated electron transfer (ET) between the heme of CDH and thiol modified gold electrodes was demonstrated using cyclic voltammetry. At low sweep rate (10 mV s^?1) and low pH (pH 4.3) up-hill ET from heme to FAD in CDH was observed. The formal potential of the heme in CDH and in the cleaved heme-subunit was found to be the same and equal to ?41 mV versus Ag ∣ AgCl at pH 5.1. The dependence of the formal potential on the pH (in the pH range 3.6–6.0) indicates the presence of one redox-linked ionisable functional group. Entropy and enthalpy changes were determined in variable temperature experiments as follows, ΔS°′=?194±14 J mol^?1 K^?1 and ΔH°′=?74±6 kJ mol^?1. The electrocatalytic behaviour of the CDH electrodes was demonstrated by addition of the enzyme substrate, cellobiose. The catalytic current was shown to decrease upon increased pH, in accordance with previous kinetic data in solution. The model of electron transport from the substrate (cellobiose) to FAD, and then through the heme domain to the electrode was confirmed in the experiments.     

Chiral resolution of R and S 1-phenylethanol on glassy carbon electrodes

The electrochemical oxidation of the chiral alcohol 1-phenyl ethanol (1PE), on carbon electrodes, using TEMPO and an achiral (luitidine) and chiral ((–)-sparteine) base was reinvestigated in order to resolve conflicting reports in literature and to ascertain whether electrochemical chiral resolution is indeed possible with this system. Investigations using electrochemical and FTIR techniques reveal that chiral oxidation occurs due to modification of the working electrode material by the chiral base. This important observation has not been reported previously, in relation to the mechanism discussed here.     

Evidence and applications for electron transfer between vitamin K3 and oxygen

In this paper we describe the electrochemical properties of vitamin K₃ (denoted VK₃) in aerated solutions, and the application potential of VK₃ in the degradation of 4-chlorophenol, 3-chloropropanl and sodium 2,3-dichloropropionate. Cyclic voltammetry revealed that the cathodic current of VK₃ decreased systematically with the addition of 3-chloropropanol and sodium 2,3-dichloropropionate. Concomitantly, a pair of new waves grew at less negative potentials. This electrode behavior suggested that VK₃ might form 1:1 complexes with 3-chloropropanol and sodium 2,3-dichloropropionate. The equilibrium constants were estimated to be about 1000 and 100 M^?l, respectively, for the reactions of VK₃ with 3-chloropropanol and sodium 2,3-dichloropropionate. Emission spectroscopy supported our postulate, although the determined equilibrium constants (10⁴ M^?l) had deviations from those obtained based on the electrochemical method. Apart from these discrepancies, VK₃ was found to be an effective catalyst for the reduction of oxygen as it was reduced. By using superoxide dismutase (SOD) and amplex red as probes, we confirmed that superoxide anion radical could be produced in aerated VK₃ solutions. In the light of these properties, attempts were made using the reduced VK₃ to degrade 4-chlorophenol, 3-chloropropanol and sodium 2,3-dichloropropionate in aerated solutions. According to the in-situ monitoring of proton levels in aerated solutions biased at ?0.80 V versus SCE, 4-chlorophenol, 3-chloropropanol and sodium 2,3-dichloropropionate could be oxygenated under the catalysis of VK₃, Fe²⁺ and SOD.     

The distribution of standard rate constants for electron transfer between thiol-modified gold electrodes and adsorbed cytochrome c

The magnitude of the total rate constant of an electrochemical reaction at small overpotentials is shown to depend on the exponentials of the distance between the electron donor and acceptor, the reorganization energy, and the formal potential of the reaction. When a population distribution of one or more of these three parameters exists, the effect on the measured standard rate constant can be illuminated by using small-amplitude a.c. electrochemical impedance spectroscopy. Assuming a Gaussian distribution of the logarithm of the standard rate constants, the results from the system of cytochrome c molecules adsorbed on thiol-modified gold electrodes yield a standard deviation of approximately 0.5 for log k°/k_avg°. The peak broadening of reversible voltammograms is related to a comparable distribution of formal potentials.     

Electrochemical reduction of the synthetic pyrethroid insecticides tralomethrin and tralocythrin at glassy carbon and mercury electrodes

Tralomethrin and tralocythrin may be electrochemically reduced in an initial irreversible two-electron process in the solvents acetonitrile and methanol at glassy carbon and mercury electrodes. The reduction potentials at mercury electrodes are between 600–900 mV less negative than at glassy carbon electrodes. Despite this difference, under both voltammetric and controlled potential electrolysis (CPE) conditions at either electrode surface, the overall reduction process for both compounds involves the elimination of two molecules of bromide ion to yield quantitatively the synthetic pyrethroid compounds deltamethrin (from tralomethrin) and cypermethrin (from tralocythrin) as products. The mechanism of bromide elimination therefore is highly dependent upon the electrode surface. At a glassy carbon electrode, reduction of the pyrethroid compounds is a concerted process involving bromide elimination. In contrast, the presence of the mercury electrode introduces the possibility of chemically reducing tralomethrin (or tralocythrin) to give deltamethrin (or cypermethrin) and a mercury bromide species at the electrode surface. The reaction provides a catalytic pathway for reduction and under voltammetric or CPE conditions the reduction process at the mercury electrode, therefore, is actually associated with the reduction of the mercury bromide species, present at the electrode surface, to elemental mercury and free bromide ion.     

DNA methylation levels of SOCS1 and LINE-1 in oral epithelial cells from aggressive periodontitis patients

Objective

DNA methylation has been shown to be critical in the regulation of inflammatory genes. Infections are able to trigger susceptibility to disease and it can be considered as potential epimutagenic factors in reshaping the epigenome. Therefore, what would be the DNA methylation status in cells present in an infected and inflamed oral environment? The aim was to verify the DNA methylation pattern in oral epithelium cells from aggressive periodontitis (AgP) patients in a specific gene involved in the inflammation control, as suppressor of cytokine signalling (SOCS)1 and in a broader way through long interspersed nuclear element (LINE)-1.

Design

Genomic DNA from oral cells of 30 generalized AgP patients and 30 healthy patients were purified and modified by sodium bisulfite. DNA methylation patterns were analyzed using combined bisulfite restriction analysis (COBRA) for SOCS1 and LINE-1.

Results

An overall scenario of demethylation was seen for both groups, whereas the healthy group presented a higher percentage of demethylation (p < 0.001), also presenting the majority of total demethylated samples (83.3% versus 70.8% in the AgP group). Total LINE-1 methylation or at each specific loci presented significant differences amongst groups.

Conclusion

Epithelial cells, present in an infected and inflamed oral environment, show different DNA methylation status from those present in a healthy oral environment, regarding the SOCS1 and LINE-1. In addition, the investigation allows detecting alterations in the DNA in a non-limited manner, since the results observed might reflect a generalized condition of the oral epithelial cells, besides reflecting the condition of the gingival epithelium cells.     

Complex electrochemistry of flavodoxin at carbon-based electrodes: results from a combination of direct electron transfer,flavin-mediated electron transfer and comproportionation

Staircase cyclic voltammetry (SCV) and differential pulse voltammetry on fully oxidized flavodoxin from Desulfovibrio vulgaris Hildenborough at the bare glassy carbon electrode give one redox couple at a potential of ?218 mV (standard hydrogen electrode (SHE)) at pH = 7.0 with an SCV peak current proportional to the scan rate. This response is caused by flavin mononucleotide (FMN), dissociated from the protein and adsorbed onto the electrode. The midpoint potential and the pK of 6.5 are equal to the values measured with free FMN in solution. When the cationic promoter neomycin is added, one additional and diffusion controlled response is observed. The midpoint potential is ?413 mV (SHE) at pH 7.0 with a redox-linked pK of 4.8 for the reduced form. The temperature dependence is ?1.86 mV K^?1, yielding ΔS° = ?179 J mol^?1 K^?1 and ΔH° = ?12.4 kJ mol^?1. Although the starting material was 100% quinone, no response was observed around the midpoint potential of the quinone to semiquinone reduction of ?113 mV (SHE) at pH 7.0, determined in an EPR-monitored titration with dithionite. Digital simulation shows that the peak currents of the second reduction couple approach a maximum value after a few cycles if comproportionation of fully reduced and fully oxidized flavodoxin occurs in solution and a small amount of semiquinone is either present initially or is generated by mediation of electrode-bound FMN. In the latter case the heterogeneous electron transfer rate between adsorbed FMN and flavodoxin is 6.3 × 10^?6 m s^?1. The implications of this anomalous behaviour for electrochemistry on flavin enzymes like glucose oxidase are discussed.     

The kinetics of mercury nucleation from Hg22+ and Hg2+ solutions on vitreous carbon electrodes

The kinetics of the electrochemical nucleation of Hg onto vitreous carbon were studied from analysis of potentiostatic current transients obtained at different overpotentials in Hg₂²⁺ and Hg²⁺ solutions. Similar sizes of critical nuclei and number densities of sites on the surface were found for nucleation of Hg from both solutions, indicating, in agreement with nucleation theory, that the rates of nucleation are controlled by surface energies and deposit–substrate interactions. Saturation number densities of nuclei were deduced from the kinetic analysis, and their relationship with those obtained from direct observation of the electrode surface is discussed. It was found that at low overpotentials, the nuclei deposited from both solutions were uniformly distributed on the electrode surface, whereas their distribution was affected by inhibition of the nucleation rates close to already established nuclei at high overpotentials. This inhibition was enhanced during deposition from Hg²⁺ solution.     

MMP-7 and TIMP-1, New Targets in Predicting Poor Wound Healing in Apical Periodontitis

Introduction

Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) are strongly associated with tissue destruction because of inflammation. In this study, we investigated the expression of MMPs and TIMPs messenger RNA and protein levels in apical periodontitis lesions.

Methods

Tissue samples from patients presenting clinical signs of chronic apical abscess (CAA) or asymptomatic apical periodontitis (AAP) were collected postoperatively and used for gene expression analysis of MMP-2, -3, -7, -9, -14, -16, and -25; TIMP-1; and TIMP-2 in real-time polymerase chain reaction. Immunohistochemistry was also performed to detect the expression of MMP-7 and TIMP-1 proteins. Lastly, U-937 cells were induced to terminal differentiation into macrophages, infected with purified Escherichia coli lipopolysaccharide, and assessed for the expression of MMP-7 and TIMP-1 using immunocytochemistry and confocal microscopy.

Results

Significantly higher messenger RNA levels were found for all genes in AAP and CAA samples when compared with healthy control samples (P < .001). AAP cases exhibited significantly higher TIMP-1 when compared with CAA cases, whereas CAA cases showed higher MMP-2, MMP-7, and MMP-9 messenger RNA levels (P < .05). We also detected positive the expression of MMP-7 and TIMP-1 proteins in the tissue samples. The expression of both MMP-7 and TIMP-1 were increased in lipopolysaccharide-stimulated cells compared with nonstimulated cells and appear to colocalize in the Golgi apparatus.

Conclusions

MMPs appear to have an influential role in CAA cases in which ongoing tissue destruction is observed. TIMPs are preferentially associated with AAP, perhaps as a subsequent defense mechanism against excessive destruction. Taken together, our findings implicate MMP and TIMP molecules in the dynamics of inflammatory periapical lesion development.     

Simultaneous determination of adenine and guanine utilizing PbO2-carbon nanotubes-ionic liquid composite film modified glassy carbon electrode

A novel and reliable electrochemical sensor based on PbO₂-carbon nanotubes-room temperature ionic liquid (i.e., 1-butyl-3-methylimidazolium hexafluorophosphate, BMIMPF6) composite film modified glassy carbon electrode (GCE) (PbO₂–MWNT–RTIL/GCE) was proposed for simultaneous and individual determination of guanine and adenine. The guanine and adenine oxidation responses were monitored by differential pulse voltammetric (DPV) measurement. Compared with the bare electrode, the PbO₂–MWNT–RTIL/GCE not only significantly enhanced the oxidation peak currents of guanine and adenine, but also lowered their oxidation overpotentials, suggesting that the synergistic effect of PbO₂, MWNT and RTIL could dramatically improve the determining sensitivity of guanine and adenine. The PbO₂–MWNT–RTIL/GCE showed good stability, high accumulation efficiency and enhanced electrocatalytic ability for the detection of guanine and adenine. Besides, the modified electrode also exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation of 0.29 V in 0.1 M pH 7.0 phosphate buffer solution (PBS). Under the optimal conditions, the detection limit for individual determination of guanine and adenine was 6.0 × 10⁻⁹ M and 3.0 × 10⁻⁸ M (S/N = 3), respectively. The proposed method for the measurements of guanine and adenine in herring sperm DNA was successfully applied with satisfactory results.     

Nonaqueous synthesis and reduction of diazonium ions (without isolation) to modify glassy carbon electrodes using mild electrografting conditions

Synthesis and reduction of diazonium cations in acetonitrile – without isolation of the solid – was carried out to modify glassy carbon (GC) electrodes with p-nitrophenyl or p-carboxyphenyl groups. Reduction of diazonium ions was investigated under a variety of conditions to further understand the process. Cyclic voltammetry at GC consistently yields two distinct peaks for the reduction of nitrobenzenediazonium ions; the first peak is particularly dependent upon the GC surface treatment and history. Subsequent reduction of the surface nitrophenyl groups was used to assess the extent of modification, and results were compared for different electrografting conditions. Electrolysis of diazonium ions, using a more positive reduction potential than typical, results in modified surfaces, and the electrolysis charge can be varied to affect the surface coverage of modifying groups, though with %RSD > 25%. Electrochemistry of common redox systems (dopamine, ruthenium hexammine, ferrocyanide) was studied as a function of modification parameters. Under mild electrografting conditions, it appears that inhomogeneously covered surfaces are produced, with blocking properties similar to spontaneously grafted GC. Additionally, electron transfer for Fe^2+/3+ is suppressed at carboxyphenyl-modified GC rather than accelerated as it is at oxidized carbon electrodes containing carbonyl groups.     

Preparation and characterization of TiO2-NTs/SnO2-Sb electrodes by electrodeposition

The novel Sb-doped SnO₂ electrodes (TiO₂-NTs/SnO₂-Sb) have been prepared by anodization, electrodeposition and annealing. TiO₂ nanotubes (TiO₂-NTs) after Sb and Sn electrodeposited were characterized using field-emission scanning electron microscopy (FE-SEM). In contrast with the traditional Sb-doped SnO₂ coating prepared by thermal decomposition, the Sb-doped SnO₂ coating prepared by electrodeposition processes show more compact. X-ray diffraction (XRD) analysis indicates that the Sb-doped SnO₂ coating prepared by electrodeposition processes are firmly combined with the TiO₂-NTs formed on the Ti substrate. Accelerated service life tests reveal that the electrodeposition processes enhance the electrochemical stability of the Sb-doped SnO₂ electrode. The cyclic voltammetry analysis shows that TiO₂-NTs/SnO₂-Sb electrodes have higher overpotential for oxygen evolution and higher electrochemical porosity. Besides, the enhanced stabilization mechanism of the TiO₂-NTs/SnO₂-Sb electrode prepared by electrodeposition processes has been studied.     

Poly[Fe(phen-NH2)3]2+ modified electrodes: proton-gated charge transfer reactions and applications in current rectification

Poly[Fe(phen-NH₂)₃]²⁺ electrodes were prepared on Au-sputtered quartz crystals (9 MHz) by oxidizing iron(II) tris(5-amino-1,10-phenanthroline) (denoted Fe(phen-NH₂)₃²⁺) complex in acetonitrile. According to the study with the electrochemical quartz crystal microbalance (EQCM) technique, the decisive step in this electrode preparation involved an anodic polymerization. The resulting polymer (denoted poly[Fe(phen-NH₂)₃]²⁺) as prepared on the electrodes showed two redox waves in aqueous solutions. One was ascribed to the electron removal from the metal center (Fe^II), poly[Fe(phen-NH₂)₃]^3+/2+, and the other one to an electron addition to the resulting polymer, poly[Fe(phen-NH₂)₃]^2+/+. For the latter waves, the peak potential shifted to more negative values in a linear manner with an increased pH. From the slope (ca. 72 mV pH^?1) and the half-height-peak width (W_1/2≈120 mV), a one-electron-one-proton transfer reaction was ascribed. Because of this property, the poly[Fe(phen-NH₂)₃]²⁺ electrode behaved as a pH sensor. The sensitivity covered a pH range from 2 to 10. Energetic probing with Fe(CN)₆^3?/4?, Ru(NH₃)₆^3+/2+ and methylviologen (MV^2+/+/0), in addition, revealed that this pH dependence could be employed to regulate the electron transfer taking place through the polyFe(phen-NH₂)₃²⁺ film. As a result, current rectifications for Fe(CN)₆^3?/4? and Ru(NH₃)₆^3+/2+ were achieved. In this study a short-range interaction between MV²⁺ and the polymeric Fe(phen-NH₂)₃²⁺ film was also found. The associated Gibbs energy change was estimated to be ?33 kJ. Long-term experiments, in addition, suggested that although a decrease in pH might deactivate the electrochemical activity of the adsorbate, the adsorbed MV²⁺ did not diffuse away from the electrode. In consequence, a vivid ‘on-off’ pattern in terms of current versus time was found during the variation in pH.     

Electrochemical response of α-H4SiW12O40 on Ag and Au electrodes

Electrochemical measurements are presented for acidic solutions containing α-H₄SiW₁₂O₄₀ adsorbed on Ag(111) and Au(111) electrodes. These measurements show that this molecule passivates the Ag surface towards solution redox events. This passivation is unique to Ag, as it is not observed on Au or carbon electrodes. Depassivation can be accomplished by moving the potential of the Ag electrode into the hydrogen evolution region. These results are discussed in terms of formation of a reduced AgSiW₁₂O₄₀ complex that is stable only at negative potentials.     

Electrochemical reduction of (1R,2r,3S,4R,5r,6S)-hexachlorocyclohexane (Lindane) at carbon cathodes in dimethylformamide

Direct reduction of Lindane (1R,2r,3S,4R,5r,6S-hexachlorocyclohexane, 1) at carbon cathodes in dimethylformamide (DMF) containing 0.10 M tetra-n-butylammonium tetrafluoroborate (TBABF₄) has been explored by means of cyclic voltammetry and controlled-potential (bulk) electrolysis. Cyclic voltammograms for reduction of 1 at a glassy carbon electrode exhibit two cathodic peaks at −1.40 V and −2.10 V as well as an anodic peak at −1.93 V; the first cathodic peak is attributed to reduction of 1 itself, whereas the second cathodic peak is due to reduction of chlorobenzene that is derived from 1. Controlled-potential (bulk) electrolyses conducted with reticulated vitreous carbon electrodes held at −1.70 or −2.20 V reveal that reduction of 1 is essentially a six-electron process that affords benzene as major product (80–100% yield) along with small amounts of chlorobenzene (3–10% yield). To account for these products, a mechanism is proposed that is supported by the results of theoretical computations based on density functional theory.     

Voltammetry in the presence of ultrasound: surface and solution processes in the sonovoltammetric reduction of nitrobenzene at glassy carbon and gold electrodes

The application of power ultrasound in electrochemistry may promote or usefully modify electrode reactions. In the work reported here the four-electron reduction of nitrobenzene in alkaline (pH 13) aqueous media was studied as a model system. The electrochemical reduction is known to follow a complex mechanism [E. Laviron, A. Vallat and R. Meunier-Prest, J. Electroanal. Chem., 379 (1994) 427], involving protonations as well as a dehydration step; both surface and solution pathways for this reduction may be observed [C. Nishihara and H. Shindo, J. Electroanal. Chem., 221 (1987) 245], depending on the nature and state of the electrode. Both under silent and ultrasonic conditions nitrobenzene is reduced on glassy carbon electrodes in a chemically reversible one-electron process followed by an irreversible three-electron reduction. At sufficiently negative potentials the reduction process remains overall four-electron, with phenylhydroxylamine as the major product even at the high current densities obtained with intense ultrasound. Glassy carbon electrodes are shown to be suitable for kinetic studies, although damage, as endorsed by an increase in roughness and capacitance and probably initiated by mechanical damage at very short electrode–horn distances, was detected by a.c. impedance, voltammetric and atomic force microscopy (AFM) methods. On gold electrodes a more complicated mechanism due to a surface reaction pathway arises. A comparison of sonovoltammetric and rotating disk voltammetric results gives evidence for the homogeneous pathway being dominant under applied ultrasound conditions. The transition between surface and solution pathways is mass flux as well as concentration dependent, and an estimate for the rate of the surface catalyzed reaction for a concentration of 1.44 mM nitrobenzene (k = 4±2 × 10^?2 cms^?1), attributed to protonation of the nitrobenzene radical anion, is obtained from combined rotating disk and sonovoltammetric data. Damage to the gold surface as monitored by various techniques is small, but manifests itself by an interesting decrease in double layer capacitance.