Current Chronopotentiometric Stripping Analysis Research Articles

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.

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.

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.

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.

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.

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.