Chronopotentiometric Stripping Analysis Research Articles

Impact of galectin-1's redox state on its lectin activity and monomer-dimer equilibrium. Focusing on oxidized Gal-1.

Galectin-1 (Gal-1) displays unique sensitivity to oxidative inactivation which appears critical in regulating its spatial and temporal activity. The two physicochemical states, i.e. monomer-dimer equilibrium and redox states, are related to Gal-1 varying functionality. In this work, we used chronopotentiometric stripping analysis, intrinsic fluorescence spectroscopy, and mobility shift assay to follow changes in the structure and lectin activity of reduced and oxidized Gal-1 forms. Our results show that monomers and dimers are similarly distributed under mild reduction and oxidation conditions. Gal-1 after its oxidation consists of at least three different monomeric forms while reduced Gal-1 only one. Lectin activity, affinity to N-acetyllactosamine, is relatively similar for low Gal-1 concentrations for both, reduced and oxidized Gal-1. However, at higher Gal-1 concentrations, we observed a ten times higher affinity for reduced than oxidized form. Further, our data indicate that the monoclonal antibodies bind preferentially to Gal-1 dimers and specifically to only some forms of its oxidized form.

Fetuin and asialofetuin at charged surfaces: Influence of sialic acid presence

• Glycoproteins, fetuin and asialofetuin, were studied at charged surfaces. • Their chronopotentiometric responses, peaks H, and impedance C - t curves differed. • Behavior of these proteins was affected by sialic acid presence in their glycan part. • Fetuin showed higher stability against electric field effects than sialofetuin. Major human diseases are associated with changes in glycosylation. The sialic acid is highly expressed saccharide residue typically terminating branches of protein glycans and glycosphingolipids. Here we compared sialoprotein fetuin with its desialylated form – asialofetuin using label-free constant current chronopotentiometric stripping (CPS) analysis and impedance C - t curves to better understand the behavior of glycoproteins at charged surfaces. Electrochemistry represents useful tool for study of molecules at charged surfaces. Using CPS peak H, we show that the terminal sialic acids presence in glycans of fetuin stabilizes its molecule against effects of electric field. Additionally the presence of negatively charged sialic acid prevents an interaction between glycoprotein and negatively charged electrode. The different behavior of the glycoproteins according to sialic acid content can also be used for easy determination of sialoglycoproteins.

Chronopotentiometric Analysis of Single Histones and Histone Octamer at Charged Surfaces

AbstractHistones play an essential role in the nucleosome structure and organization. The key role of highly positively charged histones is to stabilize negatively charged DNA inside the chromatin. Histones are basic proteins containing a large amount of electroactive residues, namely, arginine and lysine. Additionally, histone H3, on the contrary to other histones, contains cysteine residues. To understand more about the behavior of histones at charged surfaces and the influence of the electroactive amino acid residues content on the catalytic hydrogen evolution reaction, we studied histones using label‐free approaches, namely, chronopotentiometric stripping analysis (CPS) and impedance Cd–t curves. While CPS is sensitive to the structure and stability of proteins and their complexes, Cd–t curves reflect changes of the electrical double layer at the charged surface. Application of both label‐free methods appears useful for analysis of complex systems. Using a combination of these methods, we could discriminate between individual single core histones, their mixture, and their complex (octamer).

AGR2-AGR3 hetero-oligomeric complexes: Identification and characterization

In this paper we compare electrochemical behavior of two homolog proteins, namely anterior gradient 2 (AGR2) and anterior gradient 3 (AGR3), playing an important role in cancer cell biology. The slight variation in their protein structures has an impact on protein adsorption and orientation at charged surface and also enables AGR2 and AGR3 to form heterocomplexes. We confirm interaction between AGR2 and AGR3 (i) in vitro by immunochemical and constant current chronopotentiometric stripping (CPS) analysis and (ii) in vivo by bioluminescence resonance energy transfer (BRET) assay. Mutation of AGR2 in dimerization domain (E60A) prevents development of wild type AGR2 dimers and also negatively affects interaction with wild type AGR3 as shown by CPS analysis. Beside new information about AGR2 and AGR3 protein including their joint interaction, our work introduces possible applications of CPS in bioanalysis of protein complexes, including those relatively unstable, but important in the cancer research.

Influence of Protein Modification and Glycosylation in the Catalytic Hydrogen Evolution Reaction of Avidin and Neutravidin: An Electrochemical Analysis.

To investigate glycans' influence on the behavior of glycoproteins on charged surfaces, avidin and its nonglycosylated and neutralized version neutravidin were studied by label-free chronopotentiometric stripping (CPS) analysis and alternating current voltammetry combined with a mercury electrode. Despite neutravidin's and avidin's similar size and structure, their CPS responses differed due to the different amounts of catalytically active free amino groups of lysine and arginine residues. Acetylation of the proteins resulted in the suppression of their CPS responses by almost four times for avidin and by about 50 % for neutravidin, respectively. On the other hand, the presence of glycans in the acetylated avidin induced about 30 % higher chronopotentiometric response compared to the acetylated neutravidin. We suggest that the presence, size and composition of the glycans influenced the CPS signal due to differences in the orientation at a charged surface. The obtained results can be utilized in glycoprotein research.

A simple adsorptive chronopotentiometric stripping method for determination of vitamin B1 in pharmaceutical products

A new electroanalytical method for vitamin B1 determination, based on adsorptive chronopotentiometric stripping analysis and non-specific adsorption onto mercury film electrode, was developed and validated. Stripping chronopotentiograms showed a single well-defined oxidation wave corresponding to vitamin B1 at about − 0.43 V in citrate buffer pH 6.0. The most important experimental factors affecting the monitored electroanalytical response of vitamin B1 were investigated and optimised. Under the optimal experimental conditions, linear response of vitamin B1 was obtained in the concentration range of 5–50 mg dm−3, with the achieved limit of detection of 1.64 mg dm−3, and the limit of quantitation of 4.97 mg dm−3. A mean recovery of 97.1% and relative standard deviations of 3.75% were achieved. The developed electrochemical procedure was successfully applied for the determination of vitamin B1 in pharmaceutical products. The results of the proposed method were in good agreement with those obtained by parallel HPLC analyses, confirming an accuracy of the developed method.

Constant-current chronopotentiometric stripping detection of bovine serum albumin on silver amalgam particles

Highly sensitive peak H registered by constant-current chronopotentiometric stripping (CPS) analysis relates due to the catalytic hydrogen evolution reaction of proteins adsorbed on mercury-based surfaces including recently introduced silver amalgam particles (AgAPs). Since the peak H allows a structure-sensitive analysis of proteins, including those important in biomedicine and cancer research, it has been systematically studied by CPS analysis using AgAPs in this work. Conditions of AgAPs' electrodeposition on basal plane pyrolytic graphite electrode (bPGE) were optimized towards CPS analysis of model protein (bovine serum albumin, BSA) using peak H. Optimized condition for bPGE-AgAP preparation allowed sensitive CPS detection of either native or denatured BSA down to 10 nM concentration, a discrimination between both forms, and registration of the BSA thermal denaturation kinetic. Obtained results have confirmed the applicability of AgAPs in protein electroanalysis and may contribute to the further development of novel (bio)sensors or (bio)electrochemical applications using micro-volume batch, flow-through or lab-on-chip detection systems.

Interfacial properties of p53-DNA complexes containing various recognition elements

Methods which can distinguish between specific and non-specific protein interactions leading to the identification of hubs and nodes are still desired. This work shows utilization of chronopotentiometric stripping analysis in combination with a mercury electrode in the study of protein-DNA interactions at thiol-modified electrodes. The complex of tumor suppressor p53 core domain (p53CD) and DNA undergoes disintegration due to the effect of the electric field, accompanied by a remarkable increase in the electrocatalytic reduction signal. By adjusting stripping current intensities and temperature, the transition between intact and disintegrated complex reflected differences in the stabilities of sequence-specific complexes with different recognition elements. Higher stabilities of p53-DNA complexes were observed for DNA binding sites connected with cell-cycle arrest and p53 negative autoregulation, than those for DNA associated with cell apoptosis, in good concordance with electrophoretic mobility shift assay in polyacrylamide gels. These data highlight the utility of this method for studying the dynamics of surface-attached protein-DNA complexes.

Anterior gradient-3 protein-antibody interaction at charged interfaces. Label-free chronopotentiometric sensing

We developed a fast, simple, label-free method useful for the study of interactions between an antibody (Ab) and antigen based on chronopotentiometric stripping analysis and the catalytic hydrogen evolution reaction. The specific interaction of the Ab, adsorbed at a mercury electrode, with AGR3 protein induced a significant increase in chronopotentiometric peak H in comparison to both the CPS response of the Ab alone and that after incubation with nonspecifically binding proteins. Qualitatively similar results were obtained with another polyclonal antibody specific for AGR2 protein. The demonstrated results, along with previous findings indicate that the proposed technique shows promise as a new option for studying the dynamics, not only of surface-attached antibody-antigen complexes, but also other protein-protein interactions.

Electrochemical sensing of interaction of anterior gradient-2 protein with peptides at a charged interface

Anterior gradient-2 protein (AGR2) is overexpressed in many human cancers, and this protein presents a novel promising cancer biomarker. We show significant progress in understanding of the electric field effects on AGR2-peptides complexes using constant current chronopotentiometric stripping (CPS) analysis. Surface-attached AGR2-peptide complexes can be disintegrated as a result of their exposure to negative potentials. By controlling the exposure time and temperature, peaks of weakly bound non-specific complexes can be discriminated from tightly bound specific complexes. Using CPS analysis we found that mutant E60A-AGR2 forms weaker complex with peptide aptamer in comparison to wild type AGR2. These data highlight the utility of this method for studying real-time dynamics of surface-attached protein-peptide complexes.

Electrochemical behavior of sarco/endoplasmic reticulum Ca-ATPase in response to carbonylation processes

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a transmembrane protein that transfers calcium ions from the cytosol of the cell to the lumen of the sarco/endoplasmic reticulum (SR) at the expense of ATP hydrolysis during muscle relaxation. Here we report the electrochemical monitoring of a SERCA1 isoform isolated from rabbit skeletal muscle. The constant-current chronopotentiometric stripping (CPS) analysis was applied for the analysis of solubilized SERCA1 based on a catalytic hydrogen evolution reaction (CHER). Consequently we applied an optimized CPS electrochemical protocol for monitoring the interaction of SERCA1 with the dicarbonyl compounds methylglyoxal, glyoxal and 3‑deoxyglucosone. We observed that SERCA1 readily interacts with methylglyoxal at pH 7.4. Electrochemical analysis showed that 1 mg/ml (9.1 μmol/l) SERCA1 was fully modified by methylglyoxal (3 mmol/l) after 24 h. The dicarbonyl binding decreased the enzyme activity of purified SERCA1 in the following order: methylglyoxal ≫ glyoxal > 3‑deoxyglucosone. The methodology presented here could be used in further studies of the structural integrity and intermolecular interactions of membrane transporters and the study of their oxidative modifications, carbonylation or glycoxidation.

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.

Label-free chronopotentiometric glycoprofiling of prostate specific antigen using sialic acid recognizing lectins

In recent decades, it has become clear that most of human proteins are glycosylated and that protein glycosylation plays an important role in health and diseases. At present, simple, fast and inexpensive methods are sought for clinical applications and particularly for improved diagnostics of various diseases, including cancer. We propose a label- and reagent-free electrochemical method based on chronopotentiometric stripping (CPS) analysis and a hanging mercury drop electrode for the detection of interaction of sialylated protein biomarker a prostate specific antigen (PSA) with two important lectins: Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). Incubation of PSA-modified electrode with specific SNA lectin resulted in an increase of CPS peak H of the complex as compared to this peak of individual PSA. By adjusting polarization current and temperature, PSA-MAA interaction can be either eliminated or distinguished from the more abundant PSA-SNA complex. CPS data were in a good agreement with the data obtained by complementary methods, namely surface plasmon resonance and fluorescent lectin microarray. It can be anticipated that CPS will find application in glycomics and proteomics.

Chronopotentiometric sensing of anterior gradient 2 protein

The study of new proteins and particularly those involved in cancer development is still a focal point of interest. We studied the anterior gradient 2 (AGR2) oncoprotein, aberrantly expressed in a number of human cancers. For the first time the electrochemical behaviour of various variants of the AGR2 was described using constant current chronopotentiometric stripping (CPS) analysis at mercury electrodes. In the first part, we show that mutation of AGR2 protein at its sole cysteine significantly changed its CPS response compared to wild type AGR2, probably due to their different adsorption and some deviations in their structures, which were obtained by analysis of hydrogen − deuterium (H/D) exchange connected with high resolution mass spectrometry (MS). In the second part we studied the influence of His-tag modification, widely used in the purification of recombinant proteins, on CPS response and H/D exchange. Addition of a His-tag, containing positively charged and electroactive residues, affected the CPS peak H of His6-tagged AGR2 compared to non-tagged AGR2 protein due to different adsorption as well as variation in the structure at the negatively charged interface. H/D exchange MS analysis confirmed differences in the structure of these two variants even in solution. The uncovering of AGR2 behaviour at charged surfaces gives us the opportunity to study its interactions with other biomedically important proteins.

Native and denatured enzyme enterokinase determined by electrochemical methods

The aim of this work is to detect and distinguish native and denatured form of enzyme enteropeptidase. Cyclic voltammetry is usually the method of choice to be used in experiments with biomolecules or unknown chemical species. However, only hardly detectable differences between native and denatured form of enzyme enterokinase were observed using cyclic voltammetry. Therefore, chronopotentiometric stripping analysis was used for the characterization of protein conformation. Significant differences between the heights of peak H of native and denatured form of enterokinase were observed. Our experiments have proved that constant current chronopotentiometric peak H at mercury electrode is sensitive tool for the characterization of changes in protein conformation.

Chronopotentiometric sensing of specific interactions between lysozyme and the DNA aptamer

Specific DNA-protein interactions are vital for cellular life maintenance processes, such as transcriptional regulation, chromosome maintenance, replication and DNA repair, and their monitoring gives valuable information on molecular-level organization of those processes. Here, we propose a new method of label-free electrochemical sensing of sequence specific binding between the lysozyme protein and a single stranded DNA aptamer specific for lysozyme (DNAapta) that exploits the constant current chronopotentiometric stripping (CPS) analysis at modified mercury electrodes. Specific lysozyme-DNAapta binding was distinguished from nonspecific lysozyme-DNA interactions at thioglycolic acid-modified mercury electrodes, but not at the dithiothreitol-modified or bare mercury electrodes. Stability of the surface-attached lysozyme-DNAapta layer depended on the stripping current (Istr) intensity, suggesting that the integrity of the layer critically depends on the time of its exposure to negative potentials. Stabilities of different lysozyme-DNA complexes at the negatively polarized electrode surface were tested, and it was shown that structural transitions of the specific lysozyme-DNAapta complexes occur in the Istr ranges different from those observed for assemblies of lysozyme with DNA sequences capable of only nonspecific lysozyme-DNA interactions. Thus, the CPS allows distinct discrimination between specific and non-specific protein-DNA binding and provides valuable information on stability of the nucleic acid-protein interactions at the polarized interfaces.

Electrochemical sensing of concanavalin A and ovalbumin interaction in solution

In an attempt to develop a label- and reagent-free electrochemical method for the detection of lectin-glycoprotein interactions, we tested lectin-concanavalin A (ConA), glycoprotein-ovalbumin (Ova) and their complex using chronopotentiometric stripping (CPS) analysis and a hanging mercury drop electrode. Incubation of ConA with Ova resulted in an increase of the CPS peak H of the complex as compared to the CPS peaks of individual Ova and ConA proteins. Qualitatively similar results were obtained with other glycoprotein-lectin couples (ConA-RNase B and lectin from Sambucus nigra-fetuin). Using the CPS method, we were able to follow the course of complex formation in solution. Comparable responses of Ova, ConA and ConA-Ova complex were obtained not only at the mercury electrode but also with solid amalgam electrodes, which are more suitable for parallel analysis. It can be anticipated that electrochemical methods, namely CPS, will find application in glycomics and proteomics.

Electrocatalytic assay for monitoring methylglyoxal-mediated protein glycation.

Protein glycation is a complex process that plays an important role in diabetes mellitus, aging, and the regulation of protein function in general. As a result, current methodological research on proteins is focused on the development of novel approaches for investigating glycation and the possibility of monitoring its modulation and selective inhibition. In this paper, a first sensing strategy for protein glycation is proposed, based on protein electroactivity measurement. Concretely, the label-free method proposed is based on the application of a constant-current chronopotentiometric stripping (CPS) analysis at Hg-containing electrodes. The glycation process was monitored as the decrease in the electrocatalytic protein signal, peak H, observed at highly negative potentials at around -1.8 V (vs Ag/AgCl3MKCl), which was previously ascribed to a catalytic hydrogen evolution reaction (CHER). Using this method, a model protein bovine serum albumin was investigated over 3 days of incubation with the glycation agent methylglyoxal in the absence or presence of the glycation inhibitor aminoguanidine (pimagedine). The electrochemical methodology presented here could open up new possibilities in research on protein glycation and oxidative modification. The methodology developed also provides a new option for the analysis of protein intermolecular interactions using electrochemical sensors, which was demonstrated by the application of a silver solid amalgam electrode (AgSAE) for monitoring the glycation process in samples of bovine serum albumin, human serum albumin, and lysozyme.

The Chronopotentiometric Stripping Analysis of Lead Based on Abrasively Immobilized Multi-walled Carbon Nanotubes Bismuth Film Electrode

The Chronopotentiometric Stripping Analysis of Lead Based on Abrasively Immobilized Multi-walled Carbon Nanotubes Bismuth Film Electrode

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.