Teodor Adrian Enache

Boron doped diamond and glassy carbon electrodes comparative study of the oxidation behaviour of cysteine and methionine.

The electrochemical oxidation behaviour at boron doped diamond and glassy carbon electrodes of the sulphur-containing amino acids cysteine and methionine, using cyclic and differential pulse voltammetry over a wide pH range, was compared. The oxidation reactions of these amino acids are irreversible, diffusion-controlled pH dependent processes, and occur in a complex cascade mechanism. The amino acid cysteine undergoes similar three consecutive oxidation reactions at both electrodes. The first step involves the oxidation of the sulfhydryl group with radical formation, that undergoes nucleophilic attack by water to give an intermediate species that is oxidized in the second step to cysteic acid. The oxidation of the sulfhydryl group leads to a disulfide bridge between two similar cysteine moieties forming cysteine. The subsequent oxidation of cystine occurs at a higher potential, due to the strong disulfide bridge covalent bond. The electro-oxidation of methionine at a glassy carbon electrode occurs in two steps, corresponding to the formation of sulfoxide and sulfone, involving the adsorption and protonation/deprotonation of the thiol group, followed by electrochemical oxidation. Methionine undergoes a one-step oxidation reaction at boron doped diamond electrodes due to the negligible adsorption, and the oxidation also leads to the formation of methionine sulfone.

Peptide methionine sulfoxide reductase A (Msra): Direct electrochemical oxidation on carbon electrodes

The direct electrochemical behaviour of peptide methionine sulfoxide reductase A (MsrA) adsorbed on glassy carbon and boron doped diamond electrodes surface, was studied over a wide pH range by cyclic and differential pulse voltammetry. MsrA oxidation mechanism occurs in three consecutive, pH dependent steps, corresponding to the oxidation of tyrosine, tryptophan and histidine amino acid residues. At the glassy carbon electrode, the first step corresponds to the oxidation of tyrosine and tryptophan residues and occurs for the same potential. The advantage of boron doped diamond electrode was to enable the separation of tyrosine and tryptophan oxidation peaks. On the second step occurs the histidine oxidation, and on the third, at higher potentials, the second tryptophan oxidation. MsrA adsorbs on the hydrophobic carbon electrode surface preferentially through the three hydrophobic domains, C1, C2 and C3, which contain the tyrosine, tryptophan and histidine residues, and tryptophan exists only in these regions, and undergo electrochemical oxidation.

Virgin olive oil ortho-phenols—electroanalytical quantification

An electroanalytical methodology was developed for the determination of the total ortho-phenol content of virgin olive oil (VOO) with high sensitivity and reproducibility. The VOO ortho-phenol content depends on its freshness and is normally expressed as HT equivalent. Screen-printed electrodes were used with cyclic voltammetry to investigate the oxidation of catechol, phenol, hydroxytyrosol (HT), tyrosol, caffeic acid and ferulic acid. The oxidation of ortho-phenols and mono-phenols occurs following different mechanisms, and at different potentials. Using screen-printed electrodes and square wave voltammetry, an HT detection limit of 0.40 μM, was obtained. The electroanalytical methodology developed was applied to the determination of ortho-phenol content in fresh and old VOO. The HT equivalent determined for a two-year-old VOO sample was 3mg/kg, for one-year-old samples was 6-7 mg/kg, and for a fresh VOO sample 30 mg/kg, recoveries in the range of 78-93% of HT standard being obtained. The effect of VOO matrix components on the HT standard response was investigated.

Electrochemical Oxidation at a Glassy Carbon Electrode of the Anti‐Arrhythmia Drug Disopyramide

Abstract A voltammetric study of the oxidation of disopyramide has been carried out using a glassy carbon electrode. The electrochemical oxidation of disopyramide was investigated by cyclic, differential pulse, and square wave voltammetry. The oxidation of disopyramide is an irreversible, diffusion‐controlled process. The diffusion coefficient of disopyramide was calculated in pH 7.0 phosphate buffer to be D disopyramide=3.8×10−6 cm2 s−1. The oxidation of disopyramide is also pH dependent and for electrolytes with pH between 4 and 7 occurs with the transfer of one electron and one proton. In alkaline electrolytes, two consecutive charge transfer reactions are observed: both oxidation reactions involve the transfer of two electrons but only the first also involves the transfer of two protons. Two procedures for the analytical determination of disopyramide in pH 7.0 phosphate buffer were developed and compared and a detection limit LOD=1.27 µM was obtained.

Alzheimer's disease amyloid beta peptides in vitro electrochemical oxidation

The oxidative behaviour of the human amyloid beta (Aβ1-40 and Aβ1-42) peptides and a group of similar peptides: control inverse (Aβ40-1 and Aβ42-1), mutants (Aβ1-40Phe10 and Aβ1-40Nle35), rat Aβ1-40Rat, and fragments (Aβ1-28, Aβ1-16, Aβ10-20, Aβ12-28, and Aβ17-42), in solution or adsorbed, at a glassy carbon electrode, by cyclic and differential pulse voltammetry, were investigated and compared. Structurally the Aβ1-40 and Aβ1-42 sequences contain five electroactive amino acid residues, one tyrosine (Tyr10), three histidines (His6, His13 and His14) and one methionine (Met35). The Aβ peptide 3D structure influenced the exposure of the redox residues to the electrode surface and their oxidation peak currents. Depending on the amino acid sequence length and content, the Aβ peptides gave one or two oxidation peaks. The first electron transfer reaction corresponded to the tyrosine amino acid residue oxidation, and the second to both histidines and methionine amino acid residues. The highest contribution to the second oxidation peak current was from His13, followed by His14 and His6 residues, and Met35 residue had the lowest contribution. The Aβ peptides electron transfer depended on peptide hydrophobicity and 3D structure, the redox residues position in the sequence, the redox residues close to N-termini giving the highest oxidation peak currents.

Redox behaviour of verbascoside and rosmarinic acid.

The electrochemical oxidation mechanisms of rosmarinic acid (RA) and verbascoside (VB), both caffeic acid esters with two catechol moieties, were investigated. The redox mechanism is associated with the oxidation of the catechol groups, and was studied over a wide pH range by cyclic, differential pulse and square wave voltammetry, using a glassy carbon electrode. The voltammetric study revealed that both molecules, RA and VB, are reversibly oxidized in two successive pH-dependent steps each with the transfer of two electrons and two protons. Moreover, it was found that the first oxidation step is associated with the caffeic acid moiety, whereas the second oxidation step corresponds to the oxidation in VB of the hydroxytyrosol group and in RA of the 3,4-dihydroxyphenyl lactic acid residue.

Electrochemical evaluation of Abelson tyrosine-protein kinase 1 activity and inhibition by imatinib mesylate and danusertib.

Abelson tyrosine-protein kinase 1 (ABL1) catalysed phosphorylation involves the addition of a phosphate group from ATP to the tyrosine residue on the substrate abltide. The phosphorylation reactions were carried out by incubating ABL1, ATP and the substrate abltide. Adsorption at the glassy carbon electrode surface in either reaction mixtures or control solutions, followed by differential pulse voltammetry in buffer allowed detection of the variation of abltide tyrosine residue oxidation peak reflecting the occurrence of the phosphorylation reaction. The effect of abltide, ATP and ABL1 concentrations as well as the time course of the phosphorylation reaction were studied. The influence of co-adsorption of ABL1, ATP and phosphorylated abltide was evaluated and the conditions for the electrochemical detection of ABL1-catalysed phosphorylation optimised. The Michaelis-Menten constant for abltide binding KM∼4.5 μM, turnover number kcat∼11 s(-1) and enzyme efficiency kcat/KM∼2.3 s(-1) μM(-1) were calculated. The inhibition of ABL1 by imatinib mesylate and danusertib was also electrochemically investigated and IC50 values of 0.53 and 0.08 μM determined.

Electrochemical Behavior of Triflusal, Aspirin and their Metabolites at Glassy Carbon and Boron Doped Diamond Electrodes

The electrochemical behavior of triflusal (TRF) and aspirin (ASA), before and after hydrolysis in water and in alkaline medium using two different electrode surfaces, glassy carbon and boron doped diamond, was study by differential pulse voltammetry over a wide pH range. The hydrolysis products are 2-(hydroxyl)-4-(trifluoromethyl)-benzoic acid (HTB) for triflusal and salicylic acid (SA) for aspirin, which in vivo represent their main metabolites. The hydrolysis processes were also followed by spectrophotometry. The UV results showed complete hydrolysis after one hour for TRF and after two hours for ASA in alkaline solution. The glassy carbon electrode enables only indirect determination of TRF and ASA through the electrochemical detection of their hydrolysis products HTB and SA, respectively. The oxidation processes of HTB and SA are pH dependent and involve different numbers of electrons and protons. Moreover, the difference between the oxidation peak potential of SA and HTB was equal to 100 mV in the studied pH range from 1 to 8 due to the CF3 of the aromatic ring of HTB molecule. Due to its wider oxidation potential range, the boron doped diamond electrode was used to study the direct oxidation of TRF and ASA, as well as of their respective metabolites HTB and SA.

Electrochemical evaluation of glutathione S-transferase kinetic parameters

Glutathione S-transferases (GSTs), are a family of enzymes belonging to the phase II metabolism that catalyse the formation of thioether conjugates between the endogenous tripeptide glutathione and xenobiotic compounds. The voltammetric behaviour of glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione S-transferase (GST), as well as the catalytic conjugation reaction of GSH to CDNB by GST was investigated at room temperature, T=298.15K (25°C), at pH6.5, for low concentration of substrates and enzyme, using differential pulse (DP) voltammetry at a glassy carbon electrode. Only GSH can be oxidized; a sensitivity of 0.14nA/μM and a LOD of 6.4μM were obtained. The GST kinetic parameter electrochemical evaluation, in relation to its substrates, GSH and CDNB, using reciprocal Michaelis-Menten and Lineweaver-Burk double reciprocal plots, was determined. A value of KM~100μM was obtained for either GSH or CDNB, and Vmax varied between 40 and 60μmol/min per mg of GST.

Human colon adenocarcinoma HT-29 cell: Electrochemistry and nicotine stimulation

Recently, it was demonstrated that colorectal cancer HT-29 cells can secrete epinephrine (adrenaline) in an autocrine manner to auto-stimulate cellular growth by adrenoreceptors activation, and that this secretion is enhanced by nicotine, showing an indirect relation between colorectal cancer and tobacco. The electrochemical behaviour of human colon adenocarcinoma HT-29 cells from a colorectal adenocarcinoma cell line, the hormone and neurotransmitter epinephrine, and nicotine, were investigated by cyclic voltammetry, using indium tin oxide (ITO), glassy carbon (GC) and screen printed carbon (SPC) electrodes. The oxidation of the HT-29 cells, previously grown onto ITO or SPC surfaces, followed an irreversible oxidation process that involved the formation of a main oxidation product that undergoes irreversible reduction, as in the epinephrine oxidation mechanism. The effect of nicotine stimulation of the HT-29 cells was also investigated. Nicotine, at different concentration levels 1, 2 and 15 mM, was introduced in the culture medium and an increase with incubation time, 0 to 3h and 30 min, of the HT-29 cells oxidation and reduction peaks was observed. The interaction of nicotine with the HT-29 cells stimulated the epinephrine secretion causing an increase in epinephrine release concentration, and enabling the conclusion that epinephrine and nicotine play an important role in the colorectal tumour growth.