Constant Current Chronopotentiometric Stripping Analysis Research Articles

Changes of electrocatalytic response of bovine serum albumin after its methylation and acetylation

Post-translational modifications play the crucial role in biological systems and the identification of novel post-translational modifications and study of their role are gaining much attention in proteomics research. For the first time, we compared methylated and acetylated bovine serum albumin to its native and denatured form using constant current chronopotentiometric stripping analysis, phase sensitive alternating current voltammetry and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Our results showed that acetylation of bovine serum albumin resulted in decrease of chronopotentiometric peak H due to modification of electroactive residues, while predominantly non-electroactive residues were modified by methylation of serum albumin. Nevertheless, both modifications altered adsorption of protein at surface and therein influenced chronopotentiometric peak H. MALDI-TOF MS analysis confirmed modifications of serum albumin and was in good agreement with electrochemical results. The present results show the capability of label- and reagent-free electrochemical methods to detect post-translational modifications in proteins.

Investigation of protein FTT1103 electroactivity using carbon and mercury electrodes. Surface-inhibition approach for disulfide oxidoreductases using silver amalgam powder

Recently, it was shown that electrochemical methods can be used for analysis of poorly water-soluble proteins and for study of their structural changes and intermolecular (protein–ligand) interactions. In this study, we focused on complex electrochemical investigation of recombinant protein FTT1103, a disulfide oxidoreductase with structural similarity to well described DsbA proteins. This thioredoxin-like periplasmic lipoprotein plays an important role in virulence of bacteria Francisella tularensis. For electrochemical analyses, adsorptive transfer (ex situ) square-wave voltammetry with pyrolytic graphite electrode, and alternating-current voltammetry and constant-current chronopotentiometric stripping analysis with mercury electrodes, including silver solid amalgam electrode (AgSAE) were used. AgSAE was used in poorly water-soluble protein analysis for the first time. In addition to basic redox, electrocatalytic and adsorption/desorption characterization of FTT1103, electrochemical methods were also used for sensitive determination of the protein at nanomolar level and study of its interaction with surface of AgSA microparticles. Proposed electrochemical protocol and AgSA surface-inhibition approach presented here could be used in future for biochemical studies focused on proteins associated with membranes as well as on those with disulfide oxidoreductase activity.

Electrocatalysis in proteins, nucleic acids and carbohydrates

The ability of proteins to catalyze hydrogen evolution has been known for more than 80 years, but the poorly developed d.c. polarographic "pre-sodium wave" was of little analytical use. Recently, we have shown that by using constant current chronopotentiometric stripping analysis, proteins produce a well-developed peak H at hanging mercury drop and solid amalgam electrodes. Peak H sensitively reflects changes in protein structures due to protein denaturation, single amino acid exchange, etc. at the picomole level. Unmodified DNA and RNA do not yield such a peak, but they produce electrocatalytic voltammetric signals after modification with osmium tetroxide complexes with nitrogen ligands [Os(VIII)L], binding covalently to pyrimidine bases in nucleic acids. Recently, it has been shown that six-valent [Os(VI)L] complexes bind to 1,2-diols in polysaccharides and oligosaccharides, producing voltammetric responses similar to those of DNA-Os(VIII)L adducts. Electrocatalytic peaks produced by Os-modified nucleic acids, proteins (reaction with tryptophan residues) and carbohydrates are due to the catalytic hydrogen evolution, allowing determination of oligomers at the picomolar level.

Constant Current Chronopotentiometric Stripping Analysis of ‘N‐Catalyst’ in Sodium Chloride Solution and Seawater

AbstractCatalytic properties and surface activity of nitrogen containing polymeric organic material (N‐POM) were analyzed by constant current chronopotentiometric stripping analysis (CPSA) and alternating current (AC) voltammetry in sodium chloride solution (pH 8) and seawater. CPSA proved to be a suitable method for determination of low concentrations of N‐POM in seawater by measuring its ‘presodium’ catalytic peak H. A protein human serum albumin (HSA) (15% of N) was used as a model compound and the concentration of N‐POM from natural seawater samples was expressed in HSA concentration equivalents. Peak H represents an additional parameter for characterization of natural organic matter.

Electrochemistry of riboflavin-binding protein and its interaction with riboflavin

Riboflavin-binding protein (RBP, a carrier of riboflavin) plays an essential role in embryo development. Electrochemical studies of the riboflavin–RBP interactions have been so far limited to changes in polarographic and voltammetric responses of riboflavin because of lack of methods capable to detect electrochemical changes in the RBP responses. Here we used constant current chronopotentiometric stripping analysis (CPSA) with the hanging mercury drop electrode (HMDE) and square wave voltammetry (SWV) with carbon paste electrode (CPE) to investigate RBP. We found that CPSA of RBP produces electrocatalytic peak H, capable to discriminate between apoprotein and holoprotein forms of RBP. This peak is suitable for studies of RBP–riboflavin interaction at nanomolar concentrations. We observed no sign of a release of riboflavin from holoprotein adsorbed at the HMDE surface. SWV at CPE required higher concentrations of RBP and displayed almost identical oxidation peaks of apoprotein and holoprotein.

Electrochemical Determination of Thioredoxin Redox States

Thioredoxin (TRX) is a general protein disulfide reductase with a large number of biological functions, including its roles in human diseases. The TRX redox mechanism is based on reversible oxidation of two cysteine thiol groups to a disulfide, accompanied by the transfer of two protons. Using constant-current chronopotentiometric stripping analysis (CPSA) and the electrocatalytic TRX peak H, we have determined redox states of TRX at submicromolar TRX concentrations. A concentration of 1 nM TRX produces a well-developed peak H at moderate accumulation time without stirring. On the basis of this peak, interactions of 4-hydroxy-2-nonenal (HNE, product of lipid peroxidation) with TRX and the formation of TRX-HNE adducts were studied. CPSA of TRX at a carbon electrode is less sensitive and does not discriminate between reduced and oxidized forms of TRX.

Chronopotentiometric Determination of Redox States of Peptides

AbstractReduced and oxidized forms of peptides were studied by different electrochemical methods at carbon and hanging mercury drop (HMDE) electrodes. Striking differences between the reduced and oxidized peptides were obtained by constant current chronopotentiometric stripping analysis (CPSA) at HMDE. Peptides yielded electrocatalytic peak H at highly negative potentials (ca. −1.75 V). Reduced peptides adsorbed at positively charged HMDE produced substantially higher peak H than the oxidized forms. Voltammetry reflected the peptide redox state less efficiently. Different orientation of reduced and oxidized molecules at the positively charged electrode and very fast potential changes in CPSA were probably responsible for the observed effects.

Adsorptive accumulation in constant current stripping chronopotentiometry as an alternative for the electrochemical study of metal complexation by thiol-containing peptides

Abstract Constant current chronopotentiometric stripping analysis using adsorptive accumulation and negative current (AdSCP) was applied as an alternative for the study of metal complexation of glutathione (GSH) or (γ-Glu-Cys)2-Gly (PC2) with zinc ions on hanging mercury drop electrode. The behavior of the formed Zn-complexes was studied using voltammetric and chronopotentiometric methods. A multivariate curve resolution method by alternating least-squares (MCR-ALS) applied to DP voltammetric and chronopotentiometric measurements of Zn–PC2 system made possible the formulation of a complexation model. Moreover, the use of small currents in AdSCP allows one to obtain acceptable sensitivities and signal separation at very low concentrations, which is important in peptide studies under conditions similar to these of natural media.

Native and denatured bovine serum albumin. D.c. polarography, stripping voltammetry and constant current chronopotentiometry

Native and denatured states of bovine serum albumin (BSA) were studied by d.c. polarographic and voltammetric Brdicka catalytic responses (BCR) in cobalt-containing solution and by constant current chronopotentiometric stripping analysis (CPSA) in borate buffer, pH 9.3. We found that 90 nM denatured BSA produced catalytic peak H (around − 1.8 V vs. Ag/AgCl/3 M KCl). This peak was about 50-fold higher than the native protein under the same conditions. Qualitatively similar results were obtained also with other proteins in native and denatured states, such as human serum albumin, γ-globulin, myoglobin and α-crystallin. 2 nM denatured BSA produced a well-developed CPS peak (at accumulation time 5 min) while native BSA yielded almost no signal under the same conditions.

Constant Current Stripping Chronopotentiometry for the Study of Adsorbing Inert and Electrochemically Nonreversible Metal Complexes at Low Concentrations: Application to Cd and Zn Metallothioneins

Constant current chronopotentiometric stripping analysis using adsorptive accumulation and negative stripping current (AdSCP) was applied for the study of behavior of rabbit liver Cd-Zn and Zn metallothionein (Cd-Zn-MT, ZnMT) on hanging mercury drop electrode. Electrochemically inert or labile behavior of complexes can be distinguished with the application of high (1000 nA) or low (100 to 20 nA) current. Using high current, no influence of added Cd2+ or Zn2+ ions on the reduction of Cd(II) or Zn(II) complexed within MT molecule was observed, except of additions of Cd2+ to ZnMT, where bound Zn(II) was substituted by cadmium ions. With lowering of stripping current and increasing concentration of added Cd2+ or Zn2+ ions in solution progressive formation of reorganized complex with labile behavior is observed. Parallel measurement using DC voltammetry with different rates of polarization or differential pulse voltammetry were in agreement with AdSCP measurement. However, only chronopotentiometric method combines good sensitivity and signal separation at μM concentrations, inevitable in MT studies.

Sensitive Electrochemical Detection of Native and Aggregated α‐Synuclein Protein Involved in Parkinson's Disease

AbstractThe aggregation of α‐synuclein, a 14 kDa protein, is involved in several human neurodegenerative disorders, including Parkinson's disease. We studied native and in vitro aggregated α‐synuclein by circular dichroism (CD), atomic force microscopy (AFM) and electrochemical methods. We used constant current chronopotentiometric stripping analysis (CPSA) to measure hydrogen evolution catalyzed by α‐synuclein (peak H) at hanging mercury drop electrodes (HMDE) and square‐wave stripping voltammetry (SWSV) to monitor tyrosine oxidation at carbon paste electrodes (CPE). To decrease the volume of the analyte, most of the electrochemical measurements were performed by adsorptive transfer (medium exchange) from 3–6 μL drops of α‐synuclein samples. With both CPE and HMDE we observed changes in electrochemical responses of α‐synuclein corresponding to protein fibrillization detectable by CD, fluorescence and AFM. Aggregation‐induced changes in peak H at HMDE were relatively large in strongly aggregated samples, suggesting that this electrochemical signal may find use in the analysis of early stages of α‐synuclein aggregation. This assumption was documented by marked changes in the peak H potential and height in samples withdrawn at the end of the lag and the beginning of the elongation phase. Native α‐synuclein can be detected down to subnanomolar concentrations by CPSA.

Determination of Azidothymidine – an Antiproliferative and Virostatic Drug by Square‐Wave Voltammetry

AbstractThe 3′‐azido‐3′‐deoxythymidine (AZT, Zidovudine) is an antiproliferative and virostatic drug widely used in human immunodeficiency virus type 1 (HIV‐1) infection treatment. With respect to side effects of high doses and a short half‐life of AZT, a fast and simple detection method for this agent could be helpful. The aim of our study was to determine AZT levels in natural samples (urine, serum, whole blood, and cell cultures, such as the HaCaT line of keratinocytes) without their mineralization and/or purification, by means of electrochemical methods using hanging mercury drop electrode (HMDE). On this electrode, AZT undergoes irreversible reduction at the peak potential near Ep−1.1 V (vs. Ag/AgCl/3 M KCl). Reduction AZT signals were measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV), square‐wave voltammetry (SWV), and constant current chronopotentiometric stripping analysis (CPSA). In phosphate buffer (pH 8) the SWV yielded the best AZT signal with the detection limit of 1 nM. The determination of AZT concentration in biological materials is affected by electroactive components, such as proteins and DNA. For monitoring the influence of these compounds, AZT reduction was performed in the presence of 10 μg/mL calf thymus ssDNA and/or 100 μg/mL bovine serum albumin. In these cases, the detection limit increased to 0.25 μM. Also studied was the AZT concentration in keratinocyte cells (HaCaT line) during cell cultivation. It has been shown that the SWV may be considered as a useful tool for the determination of AZT concentration in cell cultures, and for monitoring AZT pharmacokinetics.

Electrochemical study of heavy metals and metallothionein in yeast Yarrowia lipolytica

The bioaccumulation of heavy metals (cadmium, nickel, cobalt and zinc) and the effect of these metals on the production of metallothionein and metallothionein-like proteins (MT) in Yarrowia lipolytica was studied by electrochemical methods. The concentrations of heavy metals were determined by differential pulse voltammetry (DPV). A combination of the constant current chronopotentiometric stripping analysis (CPSA) and adsorptive transfer stripping technique (AdTS) was used to determine the content of MT in cells. Both the bioaccumulation of heavy metals and the production of MT in different cell compartments of Y. lipolytica exposed to heavy metals were monitored. The LD 50 of each metal was determined from the number of viable cells in yeast cultures: LD 50Cd (37.5 μM), LD 50Ni (570 μM), LD 50Co (700 μM), and LD 50Zn (1800 μM). The highest concentrations of heavy metals were found in the cell wall and membrane debris while the lowest concentrations were detected in the cytoplasm. Cadmium and nickel showed the most significant effect on the production of MT. This study provides new insights into the ecophysiology of microorganisms and demonstrates the potential use of these electrochemical methods in biotechnology.

Catalytic signal of rabbit liver metallothionein on a mercury electrode: a combination of derivative chronopotentiometry with adsorptive transfer stripping

Constant current chronopotentiometric stripping analysis (CPSA) in combination with adsorptive transfer stripping (AdTS) technique was used to study the rabbit liver metallothionein (MT) on a hanging mercury drop electrode (HMDE). Metallothionein yielded a distinct, sharp chronopotentiometric signal at very negative potentials (about −1.7 V), also known as the “peak H”. The height and potential of this peak were dependent on experimental conditions, such as buffer composition, pH, and the presence oxygen in solutions. The peak H was highest in borate buffer with pH close to the isoelectric point (pI) of MT. The chronopotentiometric results contribute to a deeper understanding of the nature of catalytic hydrogen evolution and demonstrate the usefulness of the peak H in peptide and protein research.

Electrochemical DNA biosensor for the determination of benzo[ a]pyrene–DNA adducts

The metabolites of the environmental pollutant, benzo[ a]pyrene (BaP) are carcinogenic and mutagenic agents. Thus, the determination of additional products (adducts) of the interaction between DNA and BaP, attracts great interest in cancer research. In this study, the determination of interaction between BaP and calf thymus double-stranded DNA (dsDNA) was performed by using differential pulse voltammetry (DPV) and constant current chronopotentiometric stripping analysis (PSA) in connection with carbon paste electrode (CPE) or glassy carbon electrode (GCE). As a result of interaction of BaP with dsDNA, the signal obtained from the oxidation of guanine decreased and a new adduct signal at a more positive potential appeared. This new peak is attributed to the formation of an adduct from the interaction of guanine with BaP. The chemically prepared anti-7,8,9,10-tetrahydrobenzo[ a]pyrene-7,8-diol-9,10-epoxide (BPDE) adduct by using iodine oxidation was analyzed and the electrochemical signal of the adduct was observed. When the dsDNA modified GCE was immersed into various concentrations of BaP solution, the oxidation peak of guanine decreased and the adduct peak increased with the increasing BaP concentration. The partition coefficient was also obtained from the peak of BaP with dsDNA. The results revealed that the formation of adducts could be determined by using electrochemical DNA biosensors, which are fast, simple and cost-effective devices. Furthermore, this study promises that the analysis of other important adducts would benefit from the introduction of electrochemical methods.

Determination of glutathione- S-transferase traces in preparations of p53 C-terminal domain (aa320–393)

Tumor suppressor protein p53 is often expressed as a fusion protein with glutathione- S-transferase (GST). The sensitive determination of GST in p53 samples is thus necessary. We propose a method for the determination of traces of GST in the p53 C-terminus based on the constant current chronopotentiometric stripping analysis (CPSA) with hanging mercury drop electrode (HMDE). GST produces a catalytic signal in cobalt-containing solutions due to cysteine residues. A large excess of the C-terminus does interfere with the determination because of the lack of cysteines in the molecule. This method is simple and very sensitive and is capable of detecting <1% GST in the p53 sample.

Interactions of antitumor drug daunomycin with DNA in solution and at the surface

The interaction of the antitumor drug daunomycin with double-stranded (ds) calf thymus DNA was studied in solution and at the electrode surface by means of cyclic voltammetry and particularly by constant-current chronopotentiometric stripping analysis (CPSA) with the carbon paste electrodes (CPE). As a result of intercalation of this drug between the base pairs in dsDNA, the CPSA daunomycin peak δ decreased and a new more positive shoulder δ b appeared. This shoulder was attributed to the oxidation of the drug intercalated in DNA. Under the same conditions almost no changes in the DNA peak G ox (due to oxidation of guanine residues) were observed. It was shown that daunomycin adheres strongly to the bare CPE (resisting washing) so that a daunomycin-modified electrode can be easily prepared. Daunomycin immobilized at CPE interacted with DNA on immersion of the modified electrode into the dsDNA solution, showing a decrease of peak δ and a well-separated peak δ b instead of the shoulder (which resulted from the interaction of DNA with daunomycin in solution). When the DNA-modified CPE was immersed into a daunomycin solution the peak G ox increased in dependence on daunomycin concentration or on the time of interaction of daunomycin with dsDNA at the electrode surface. Such changes in peak G ox were observed only at submicromolar concentrations of daunomycin. At higher daunomycin concentrations or at longer interaction time intervals a daunomycin peak appeared, which was substantially smaller and more positive than the peak of free daunomycin. The increase of the DNA peak G ox was attributed to interaction of daunomycin from the side of the DNA double helix not contacting the electrode surface. Such binding may induce changes in the DNA structure including bending of the DNA molecule which may result in the increase of peak G ox. Our results thus suggest that the interaction of daunomycin with DNA anchored at the surface may significantly differ from that with DNA in solution. The prospects of using of electroanalytical methods in studies of DNA–drug interactions are discussed.

Chronopotentiometric stripping of DNA at mercury electrodes

AbstractDenatured (single‐stranded, ss) and native (double‐stranded, ds) DNAs were studied by constant current chronopotentiometric stripping analysis (CPSA) at a hanging mercury drop electrode (HMDE). In agreement with the previous voltammetric studies at neutral pH values ssDNA produced CPS cathodic peak CA (due to reduction of cytosine and adenine) and anodic peak G (due to oxidation of the guanine reduction product) at dt/dE against E curves. The same peaks produced by dsDNA were substantially smaller. Differences in heights (areas) of peaks G and CA produced by ss and dsDNAs at partial and full electrode coverage are compared and discussed. Using CPSA a well‐developed and symmetrical peak CA was obtained at low DNA concentrations compared to a poorly measurable inflection produced by differential pulse or linear sweep voltammetries under the same conditions. The CPS peak CA can be used also in adsorptive transfer stripping (AdTS, medium exchange) experiments. In the previous AdTS voltammetric measurements only peak G was exploited while the voltammetric peak CA was poorly defined to be of any use in combination with DNA‐modified electrodes. CPS peaks were measured in the presence and in absence of air. It was shown that in the presence of air peak CA is enhanced while peak G decreased. We compared the CPSA of ssDNA at the mercury and carbon paste electrodes (CPE). The CPS peak G (obtained with HMDE) seems to be the best choice for the electrochemical determination of DNA in highly diluted solution. With 10 min adsorptive preconcentration a detection limit below 20 ng of ssDNA per mL was obtained at HMDE. At CPE the limit of detection (of the same ssDNA) was at least two‐fold higher.

Trace measurements of plasmid DNAs by adsorptive stripping potentiometry at carbon paste electrodes

Supercoiled, linear and denatured forms of plasmid pUC19 DNA can be adsorbed at carbon paste (CPE) and pyrolytic graphite (PGE) electrodes and detected by constant current chronopotentiometric stripping analysis (PSA) at low bulk concentrations. The determination is based on the PSA peak at about 1.0 V (against Ag|AgCl) due to the oxidation of guanine residues in the DNA. In agreement with earlier voltammetric data obtained with chromosomal DNAs at carbon electrodes, the denatured DNA produces a higher signal than linear and supercoiled DNAs. Compared with voltammetry, the sensitivity of PSA is higher by several orders of magnitude. PSA detection limits (obtained with a CPE) are about 100 ng ml −1 for supercoiled and linear DNAs and 15 ng ml −1 for denatured DNA. This great increase in sensitivity of the analysis at carbon electrodes offers new possibilities for electrochemical research, involving various forms of plasmid DNA.