Electrochemical behaviour of proteins at graphite electrodes. II. Electrooxidation of amino acids

Electrochemical oxidation of L,alpha-amino acids at a paraffin-wax impregnated spectroscopic graphite electrode (WISGE) was studied by means of linear sweep, cyclic, phase-sensitive alternating current and differential pulse voltammetric techniques. It was found that out of the amino acids usually occurring in proteins only tyrosine, tryptophan, histidine, cystine, cysteine and methionine were oxidized at the WISGE. At relatively low concentrations of amino acids (up to ca. 2 x 10(-4) M) the electrode process in which the amino acids are oxidized at the WISGE has the characteristics of an irreversible reaction controlled by diffusion. Coulometric measurements showed that oxidation of tyrosine and tryptophan at the WISGE, i.e. of amino acids which are responsible for the oxidizability of proteins at graphite electrodes, is a two-electron process. At higher concentrations of tyrosine-and tryptophan (above ca. 2 x 10(-4) M) adsorption of the oxidation product of these amino adds was demonstrated.     

Some observations on the periodate oxidation of amino compounds

Various aliphatic and aromatic amines are oxidized by sodium metaperiodate and these reactions have been studied quantitatively in acidic, unbuffered and basic media. Significant differences have been observed between the behaviour of aliphatic and aromatic amines. Certain compounds also behaved differently under acidic and basic conditions. These reactions are related to the periodate oxidation of amino acids and, from observations on a number of glycine derivatives, a reaction mechanism is proposed for this process.     

Multi-channel electrochemiluminescence of luminol at a copper electrode.

Multi-channel electrochemiluminescence (ECL) of luminol at a copper electrode has been studied under conventional cyclic voltammetric (CV) conditions. Compared with the ECL of luminol at other electrodes, three ECL peaks were observed at 0.30, -0.24 and -0.65 V (vs. SCE), respectively, which was also imaged by a CCD camera. The effects of potential scan direction, anodic reverse potential, the presence of N2 and O2 of the solution, the pH of the solution, the NaNO3 concentration and the potential scan rate were examined. The effect of n-alkanethiol self-assembled monolayers on copper electrodes and 20 L-amino acids, dopamine, adrenaline and noradrenaline on the ECL of luminol were also investigated. The emission spectra of various ECL peaks at different potentials demonstrated that all ECL peaks were related to the luminol reaction. The results show that the oxygen dissolved in solution and copper oxide covered on the surface of the electrode play an important role in the luminol ECL process at a copper electrode. It has been proposed that three ECL channels of luminol at a copper electrode resulted from the reactions of luminol or luminol radical electrooxidized by luminol with various electrogenerated oxygen-containing species, such as O2, OOH- and copper oxides at different potentials.     

Differences in the catalytic properties of fragments of the ceruloplasmin molecule

Human ceruloplasmin (Cp) molecule is split into fragments by a contaminating protease upon storage of enzyme preparations. These fragments were separated by SDSPAAG electrophoresis and their Mr were estimated. Separate fragments were subjected to immunoelectrophoresis in agarose gel containing rabbit antibodies to human Cp. The immunoprecipitation peaks were then specifically stained to reveal the oxidase activity of the fragments towards o-dianisidine and L-cysteine. All the fragments were able to oxidize the latter, however, only the whole Cp molecule and the two of its largest fragments could oxidize the former. It seems likely that oxidation of L-cysteine does not require the presence of several copper ions constituting the catalytic centre of the blue oxidase (Cp.). contrarily, o-dianisidine seems to be oxidized by the multicopper active site of the enzyme rather than by the autonomously acting singular copper(s). Since o-dianisidine is oxidized by the fragments of Cp lacking the C-terminal polypeptide, which was thought to bind all the coppers of the active centre, it was assumed that some of the latter are bound by amino acids located in another part of the molecule.     

An electrocatalytic transducer for l-cysteine detection based on cobalt hexacyanoferrate nanoparticles with a core–shell structure

The electrocatalytic oxidation of l-cysteine (CySH) was studied on cobalt hexacyanoferrate nanoparticles with a core–shell structure (iron(III) oxide core–cobalt hexacyanoferrate shell) using cyclic voltammetry and chronoamperometry. Voltammetric studies represented two quasi-reversible redox transitions for the nanoparticles in phosphate buffer solution (pH 7.4). In the presence of CySH, the anodic peak current of the Fe(II)/Fe(III) transition was increased, followed by a decrease in the corresponding cathodic peak current, whereas the peak currents related to the Co(II)/Co(III) transition almost remained unchanged. The results indicated that the nanoparticles oxidized CySH via a surface mediation electrocatalytic mechanism. The catalytic rate constant, the electron transfer coefficient, and the diffusion coefficient involved in the electrooxidation process of CySH are reported here. Ultrasensitive and time-saving determination procedures were developed for the analysis of the CySH, and the corresponding analytical parameters are reported. According to the proposed methods, CySH was determined with detection limits of 40 and 20 nm in batch and flow systems, respectively. The proposed amperometric method was also applied to the analysis of CySH in human urine and serum blood samples.     

Electrochemistry of neuropeptides: a possible method for assay and in vivo detection

The electrochemical activity of catechol and indoleamines has been utilized to develop sensitive assays for amine neuro-transmitters and metabolites. We present evidence that several neuropeptides are electrochemically active and that this property may be used to develop peptide assays similar in sensitivity to those available for the amines. The electrochemical activity of twenty synthetic neuropeptides and their constituent amino acids were tested in buffered solutions using differential pulse voltammetry (DPV) and micro-carbon paste working electrodes. The studies show that peptides which are electroactive include Met-and Leu-enkephalin, the cholecystokinins (CCK-8, CCK-4), caerulein, neurotensin, gonadotrophin releasing hormone (LH-RH), α-melanocyte stimulating hormone (αMSH), somatostatin and vasopressin. The electrochemical oxidation potentials of these peptides are distinct and separable from those of the amines and are apparently associated with the presence and combination of specific amino acids (tyrosine, tryptophan and cysteine) in the peptide sequence. DPV $\text{in vitro}$ was used to demonstrate the presence of 5-hydroxytryptamine and caerulein in extracts of amphibian skin. With electrodes implanted in the rat striatum DPV $\text{in vivo}$ exhibited oxidation potentials which may relate to neuropeptide oxidation.     

Electrochemical reactions of horse heart cytochrome c at graphite electrodes

The interaction of cytochrome c with a paraffin-wax-impregnated spectroscopic graphite electrode (WISGE) was studied in a medium consisting of 0.1 M potassium phosphate, pH 7.0, by means of differential pulse and cyclic voltammetry. Ferricytochrome c yields on voltammograms a single cathodic peak C around a potential of -0.3 V (vs. Ag/AgCl) and two anodic peaks AI and AII around the potentials of 0.66 and 0.89 V, respectively. Cathodic peak C corresponds to a catalytic reaction during which ferricytochrome c is reduced to ferrocytochrome c: ferricytochrome c is then regenerated by chemical oxidation of ferrocytochrome c by oxygen adsorbed at the WISGE surface. The first, more negative anodic peak AI corresponds to anodic electrochemical oxidation of tyrosine residues, whereas the second, more positive anodic peak (peak AII) corresponds to an anodic reaction of haemin. Voltammetry at a WISGE may provide a valuable technique for obtaining data about cytochrome c properties on electrically charged surface.     

Electrochemical behaviors of amino acids at multiwall carbon nanotubes and Cu2O modified carbon paste electrode

A carbon paste electrode modified with multiwall carbon nanotubes and copper(I) oxide (MWCNT-Cu₂O CPME) was fabricated, and the electrochemical behaviors of 19 kinds of natural amino acids at this modified electrode were studied. The experimental results showed that the various kinds of amino acids without any derivatization displayed obvious oxidation current responses at the modified electrode. It was also found that the current response values of amino acids were dependent mainly on pH values of buffer solutions. The phenomenon could be explained by the fact that the amino acids suffered complexation or electrocatalytic oxidation processes under different pH values. Six kinds of amino acids (arginine, tryptophan, histidine, threonine, serine, and tyrosine), which performed high-oxidation current responses in alkaline buffers, were selected to be detected simultaneously by capillary zone electrophoresis coupled with amperometric detection (CZE-AD). These amino acids could be perfectly separated within 20 min, and their detection limits were as low as 10⁻⁷ or 10⁻⁸ mol L⁻¹ magnitude (signal/noise ratio = 3). The above results demonstrated that MWCNT-Cu₂O CPME could be successfully employed as an electrochemical sensor for amino acids with some advantages of convenient preparation, high sensitivity, and good repeatability.     

Cobalt hydroxide nanoparticles modified glassy carbon electrode as a biosensor for electrooxidation and determination of some amino acids

The electrochemical behavior of some amino acids was investigated on cobalt hydroxide nanoparticles modified glassy carbon (CHM-GC) electrode in alkaline solution. The process of oxidation and its kinetics were established by using cyclic voltammetry, chronoamperometry techniques, and steady-state polarization measurements. The results revealed that cobalt hydroxide promotes the rate of oxidation by increasing the peak current, so these bimolecular reactions are oxidized at lower potentials. Cyclic voltammograms and chronoamperometry indicate a catalytic EC′ mechanism to be operative with electrogeneration of Co(IV) as the electrochemical process. Also, the process is diffusion controlled and the current-time responses follow Cottrellian behavior. This result was confirmed by steady-state measurements. The rate constants of the catalytic oxidation of amino acids and the electron transfer coefficients are reported.     

Efficient electrocatalysis of L-cysteine oxidation at carbon ionic liquid electrode

The electrochemistry of L-cysteine (CySH) in neutral aqueous media was investigated using carbon ionic liquid electrode (CILE). Comparative experiments were carried out using glassy carbon electrodes. At CILE, highly reproducible and well-defined cyclic voltammograms were obtained for l-cysteine with a peak potential of 0.49V vs Ag/AgCl, showing that CILE manifests a good electrocatalytic activity toward oxidation of l-cysteine. A linear dynamic range of 2-210microM with an experimental detection limit of 2microM was obtained. The method was successfully applied to the determination of l-cysteine in a sample of soya milk. Cysteine oxidation at CILE does not result in deactivation of the electrode surface. Mechanistic studies showed that, at CILE, the overall CySH oxidation is controlled by the oxidation of the CyS(-) electroactive species.     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.     

Nitrite and nitric oxide treatment of Helix pomatia hemocyanin: single and double oxidation of the active site.

The reaction of nitrite and nitric oxide with Helix pomatia hemocyanin has been studied. One or both of the two copper ions in the active site can be oxidized, depending upon reaction conditions. The single oxidation of the oxygen binding site can be reversed by reduction with hydroxylamine, and the oxygen binding properties of the protein are simultaneously restored. The experiments, including electron paramagnetic resonance, indicate that nitric oxide is not a ligand of copper in the singly oxidized active site and that the oxidized copper ions is coupled to at least two nitrogen atoms of amino acid residues. The doubly oxidized protein can be reduced to a singly oxidized one with ascorbic acid or hydroxylamine; the latter reagent is again able to reduce the singly oxidized state and to restore the oxygen binding properties.     

Bionanotechnology approach for FAD-dependent enzymes with nanomaterials sensor

We have reported the modification of biomolecule with nanomaterials. In this paper, the electrochemical response of different FAD-dependent enzymes at carbon nanomaterials modified electrode. The modified electrode also exhibits a promising enhanced electrocatalytic activity toward the oxidation of substrate. Different methods were used for fabrication of modified electrode. The presence of nanomaterials enhances the enzyme loading and stability. Cyclic voltammograms (CVs) were used for the determination of substrate and the apparent coefficient values for these compounds at different electrodes. Finally, we have studied the surface morphology of the modified electrode using scanning electron microscopy (SEM), which revealed that enzyme is coated on nanomaterials.     

Direct voltammetry and electrocatalytic properties of catalase incorporated in polyacrylamide hydrogel films

The direct voltammetry and electrocatalytic properties of catalase (Cat) in polyacrylamide (PAM) hydrogel films cast on pyrolytic graphite (PG) electrodes were investigated. Cat-PAM film electrodes showed a pair of well-defined and nearly reversible cyclic voltammetry peaks for Cat Fe(III)/Fe(II) redox couples at approximately -0.46 V vs. SCE in pH 7.0 buffers. The electron transfer between catalase and PG electrodes was greatly facilitated in the microenvironment of PAM films. The apparent heterogeneous electron transfer rate constant (k(s)) and formal potential (E degrees ') were estimated by fitting square wave voltammograms with non-linear regression analysis. The formal potential of Cat Fe(III)/Fe(II) couples in PAM films had a linear relationship with pH between pH 4.0 and 9.0 with a slope of -56 mV pH(-1), suggesting that one proton is coupled with single-electron transfer for each heme group of catalase in the electrode reaction. UV-Vis absorption spectroscopy demonstrated that catalase retained a near native conformation in PAM films at medium pH. The embedded catalase in PAM films showed the electrocatalytic activity toward dioxygen and hydrogen peroxide. Possible mechanism of catalytic reduction of H(2)O(2) at Cat-PAM film electrodes was proposed.     

Synthesis and bioelectrochemical behavior of aromatic amines

Four aromatic amines 1-amino-4-phenoxybenzene (A₁), 4-(4-aminophenyloxy) biphenyl (A₂), 1-(4-aminophenoxy) naphthalene (A₃) and 2-(4-aminophenoxy) naphthalene (A₄) were synthesized and characterized by elemental, spectroscopic (FTIR, NMR), mass spectrometric and single crystal X-ray diffraction methods. The compounds crystallized in monoclinic crystal system with space group P2₁. Intermolecular hydrogen bonds were observed between the amine group and amine/ether acceptors of neighboring molecules. Electrochemical investigations were done using cyclic voltammetry (CV), square wave voltammetry (SWV) and differential pulse voltammetry (DPV). CV studies showed that oxidation of aromatic amines takes place at about 0.9 V (vs. Ag/AgCl) and the electron transfer (ET) process has irreversible nature. After first scan reactive intermediate were generated electrochemically and some other cathodic and anodic peaks also appeared in the succeeding scans. DPV study revealed that ET process is accompanied by one electron. DNA binding study of aromatic amines was performed by CV and UV–visible spectroscopy. These investigations revealed groove binding mode of interaction of aromatic amines with DNA.     

Molecular "wiring" glucose oxidase in supramolecular architecture

Supramolecular organized multilayers were constructed by multiwalled carbon nanotubes modified with ferrocene-derivatized poly(allylamine) redox polymer and glucose oxidase by electrostatic self-assembly. From the analysis of voltammetric signals and fluorescence results, a linear increment of the coverage of enzyme per bilayer was estimated, which demonstrated that the multilayer is constructed in a spatially ordered manner. The cyclic voltammograms obtained from the indium tin oxide (ITO) electrodes coated by the (Fc-PAH@CNT/GOx)n multilayers revealed that bioelectrocatalytic response is directly correlated to the number of deposited bilayers; that is, the sensitivity is tunable by controlling the number of bilayers associated with ITO electrodes. The incorporation of redox-polymer-functionalized carbon nanotubes (CNT) into enzyme films resulted in a 6-10-fold increase in the glucose electrocatalytic current; the bimolecular rate constant of FADH2 oxidation (wiring efficiency) was increased up to 12-fold. Impedance spectroscopy data have yielded the electron diffusion coefficient (De) of this nanostructure to be over 10(-8) cm2 s(-1), which is typically higher than those systems without CNT by at least a factor of 10, indicating that electron transport in the new supramolecular architecture was enhanced by communication of the redox active site of enzyme, redox polymer, and CNT.     

Tea catechins inhibit cholesterol oxidation accompanying oxidation of low density lipoprotein in vitro

Endogenous oxidized cholesterols are potent atherogenic agents. Therefore, the antioxidative effects of green tea catechins (GTC) against cholesterol oxidation were examined in an in vitro lipoprotein oxidation system. The antioxidative potency of GTC against copper catalyzed LDL oxidation was in the decreasing order (-)-epigalocatechin gallate (EGCG)=(-)-epicatechin gallate (ECG)>(-)-epicatechin (EC)=(+)-catechin (C)>(-)-epigallocatechin (EGC). Reflecting these activities, both EGCG (74%) and ECG (70%) inhibited the formation of oxidized cholesterol, as well as the decrease of linoleic and arachidonic acids, in copper catalyzed LDL oxidation. The formation of oxidized cholesterol in 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation of rat plasma was also inhibited when the rats were given diets containing 0.5% ECG or EGCG. In addition, EGCG and ECG highly inhibited oxygen consumption and formation of conjugated dienes in AAPH-mediated linoleic acid peroxidative reaction. These two species of catechin also markedly lowered the generation of hydroxyl radical and superoxide anion. Thus, GTC, especially ECG and EGCG, seem to inhibit cholesterol oxidation in LDL by combination of interference with PUFA oxidation, the reduction and scavenging of copper ion, hydroxyl radical generated from peroxidation of PUFA and superoxide anion.     

A technique of comparative amperometric measurements for identification of released neurotransmitters

A recently developed technique for electrochemical identification opens new prospects for stydying transmitter release from single cells. Using an express cyclic voltammetric technique, we studied the properties of two neurontransmitters, serotonin and dopamine, which can be simultaneously released in the course of synaptic transmission. We showed that both transmitters determine the presence of an oxidation peak and a reduction peak. Only the amplitudes of the reduction peaks for dopamine and serotonin were significantly different: the reduction peak for dopamine was 3.65 times higher than that for serotonin, while the oxidation peaks showed no significant difference. We also showed that positions of the peaks along the potential scale also can be used for differentiation of the substances. The electrode potentials for the maximum serotonin and dopamine reduction peaks were significantly different, and the oxidation peak for dopamine was shifted by 125 mV toward higher potentials. Testing of a solution with both neurotransmitters added showed that an integral voltammogram is the result of simple summation of two separate “gage” voltammograms for serotonin and dopamine. This finding shows that the tested agents do not interact. Thus, the “gage” voltammograms can be used in electrophysiological experiments for identification of the released compound types.     

The IH-NMR-study of the copper surrounding in plantacyanin

The 300-MHz proton NMR spectra of oxidized, reduced and apo-forms of plantacyanin were studied. The data obtained show that one of two histidines is far from copper whereas the other is a ligand of the metal. Ligands of copper are also two methionines and, possibly, tryptophan. Although the surrounding of copper in plastocyanin consists of two sulfur and two nitrogen atoms, only histidine and methionine are invariant ligand amino acids of the metal in these two copper proteins from plants.     

Amine Oxidases of Marine Phytoplankton

Some phytoplankton utilized a novel mechanism for obtaining nitrogen from primary amines. They oxidized the primary amines to produce extracellular hydrogen peroxide and aldehydes and used the third reaction product, ammonium, as a nitrogen source. The specificity, regulation, inhibition by bromoethylamine, and potential dependence on copper of this process are described.