Current Chronopotentiometric Stripping Research Articles

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.

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.

Effects of ex situ chronopotentiometric analysis on stability of bovine serum albumin on mercury electrodes

Constant current chronopotentiometric stripping (CPS) allows analysis of proteins based on measured peak H resulting from catalytic hydrogen evolution reaction. The technique is label-free and sensitive to the structure and stability of the protein adsorbed at the mercury electrode. Comparison of proteins must be carried out under the same conditions, including ionic strength, temperature, pH, and accumulation potential and time, as all of these factors can affect the protein stability and structure on the electrode surface. Here we show that for bovine serum albumin, uncontrolled disconnection of the cell after accumulation, as is necessary for ex situ CPS measurements, can cause an increased susceptibility to electric field-induced denaturation during the subsequent CPS measurement. This destabilization is attributed to oxidation of the Hg electrode during disconnection. For this reason, care much be taken when ex situ CPS measurement of protein is performed.

BSA‐Polysaccharide Interactions at Negatively Charged Electrode Surface. Effects of Current Density.

AbstractConstant current chronopotentiometric stripping (CPS) peak H due to catalytic hydrogen evolution reaction on Hg‐containing electrodes appeared useful in the analysis of protein complexes with single‐stranded and double‐stranded DNA as well as with peptides. In dependence on stripping current (Istr), structural transition of the protein alone or in complexes can be followed as a result of the protein exposure to electric field effects. For the first time we show here that the CPS analysis can be used for the study of the interaction of BSA with a polysaccharide namely sodium alginate (SA). BSA‐SA complex formation was accompanied by the shift of the structural transition of BSA to lower ‐Istr intensities. Another polysaccharide dextran did not alter Istr‐dependent structural transition of BSA. BSA‐SA complex can be disturbed by an electric field effect or high ionic strength confirming the electrostatic nature of BSA‐SA interaction.

Chronopotentiometric Analysis of Proteins at Charged Electrode Surfaces

AbstractProtein properties and functions are strongly dependent on the structure and amino acid content. In this work, catalytic hydrogen evolution reaction (CHER) of five proteins (human serum albumin, lysozyme, β‐synuclein, H2 A and H3 histones) were studied using constant current chronopotentiometric stripping (CPS) with the aim to find out the association between protein content and its electrochemical response. We have shown that the height and potential of CPS peak H in dependence on accumulation potential differed for the studied proteins, while the CPS peak area was almost the same for all of them. CV and CPS peaks H of Cys‐containing proteins appeared at less negative potentials in comparison to proteins without Cys, suggesting easier CHER. Acidic and basic proteins not containing Cys can be also recognized due to their different CPS response after their adsorption at the positive and negative charged interface.

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.

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.

Electrochemical analysis of Os(VI)-modified glycoproteins and label-free glycoprotein detection eluted from lectin capillary column

Earlier we showed that using Os(VI)temed complex, glycans can be modified directly in glycoproteins and detected voltammetrically at μM concentrations at carbon electrodes. Here we used Os(VI)2,2′-bipyridine for modification of ribonucleases and Hg electrodes for voltammetric detection. We show that glycosylated RNase B produced electrocatalytic signal (after separation from the reaction mixture) at pM concentrations while non-glycosylated RNase A yielded negligible signal under the same conditions. Using Os(VI)temed and carbon electrodes voltammetric detection was less sensitive but allowed detection of RNase B in an excess of non-glycosylated protein, directly in the reaction mixture. We also showed that the constant current chronopotentiometric stripping (CPS) peak H (which is due to the ability of some amino acid residues in proteins to catalyze hydrogen evolution at Hg electrodes) could be used for protein structure-sensitive analysis at mercury electrodes. Using this peak, here we show that glycosylation of RNase stabilizes its molecule at the electrode. On the other hand, Os(VI)temed modification results in destabilization of this surface-attached protein. Peak H was also used for detection of RNase B separated from a large excess of non-glycosylated proteins on a lectin (concanavalin A) monolithic flow-through column.

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.

Voltammetric and chronopotentiometric protein structure-sensitive analysis

Previously we showed that constant current chronopotentiometric stripping (CPS) is convenient for protein analysis based on the ability of some amino acid residues to catalyze hydrogen evolution on mercury-containing electrodes. This method showed a remarkable sensitivity to changes in protein structures, including protein denaturation and even small protein damage. Here we used normal pulse voltammetric stripping (NPVS) with bare and dithiothreitol-modified hanging mercury drop electrode. We found that NPV pulses denatured the surface-attached protein but we showed conditions under which this method was able to distinguish native and denatured proteins with sensitivity approaching that of CPS. Using NPVS it was possible to follow bovine serum albumin (BSA) thermal denaturation as well as to investigate the effect of NPV pulses on the structure of the surface-attached protein. In addition to BSA we studied several proteins, such as human serum albumin, ovalbumin, urease, aldolase, concanavalin A, histone and vasopressin peptide. Our results suggest that CPS remains the method-of-choice for studies of changes in protein structure and of biochemical processes, such as DNA-protein specific binding, lectin-glycoprotein interactions, detection of protein damage, etc., while voltammetric methods, such as NPVS may suit better for investigation of the processes which proteins undergo at the electrode surface.

Protein Structure‐sensitive Analysis by Normal Pulse Voltammetry

AbstractEarlier we showed that constant current chronopotentiometric stripping (CPS) at mercury‐containing electrodes reflects changes in proteins, such as denaturation, single amino acid exchange or protein damage by physical and chemical agents. Here we attempted to compare performance of the galvanostatic CPS with the potentiostatic normal pulse voltammetry (NPV). We have found that repeated 50 ms or longer NPV pulses denature the surface‐attached protein. Using shorter pulses and adapting other NPV settings we have been able to obtain a good resolution of native (folded) and denatured (unfolded) bovine serum albumin. On the other hand, partial denaturation of the surface‐attached native bovine serum albumin during the potential scanning may prevent detection of small conformational changes in the protein.

Protein structural transition at negatively charged electrode surfaces. Effects of temperature and current density

Earlier it was shown that surface-attached bovine serum albumin undergoes ionic strength-induced structural transition at mercury electrodes. Using constant current chronopotentiometric stripping (CPS) here we show that this electric field-driven transition is influenced also by other factors, such as temperature, current density, and particularly by the time of surface-attached protein exposure to the negative potentials. For example, 1s exposure to –1.6V (against Ag/AgCl/3M KCl) results in the protein denaturation. Exposures longer than 1s resulted in protein denaturation even at less negative potentials. On the other hand, in CPS much shorter exposure times can be used. Exposing the surface-attached protein to negative potentials for ms time intervals the protein stability can be tested at different current densities. We investigated several proteins such as bovine and human serum albumins, ovalbumin, α-macroglobulin and α-synuclein, which showed various profiles in dependence on different parameters, including time of the protein exposure to negative potentials, ionic strengths and temperature. We believe that using this approach, new methods for testing surface-attached protein stabilities in relation to the electric field effects and to other factors can be developed.

Electrochemical sensing of tumor suppressor protein p53–deoxyribonucleic acid complex stability at an electrified interface

Electrochemical biosensors have the unique ability to convert biological events directly into electrical signals suitable for parallel analysis. Here we utilize specific properties of constant current chronopotentiometric stripping (CPS) in the analysis of protein and DNA–protein complex nanolayers. Rapid potential changes at high negative current intensities (Istr) in CPS are utilized in the analysis of DNA–protein interactions at thiol-modified mercury electrodes. P53 core domain (p53CD) sequence-specific binding to DNA results in a striking decrease in the electrocatalytic signal of free p53. This decrease is related to changes in the accessibility of the electroactive amino acid residues in the p53CD–DNA complex. By adjusting Istr and temperature, weaker non-specific binding can be eliminated or distinguished from the sequence-specific binding. The method also reflects differences in the stabilities of different sequence-specific complexes, including those containing spacers between half-sites of the DNA consensus sequence. The high resolving power of this method is based on the disintegration of the p53CD–DNA complex by the electric field effects at a negatively charged surface and fine adjustment of the millisecond time intervals for which the complex is exposed to these effects. Picomole amounts of p53 proteins and DNA were used for the analysis at full electrode coverage but we show that even 10–20-fold smaller amounts can be analyzed. Our method cannot however take advantage of very low detection limits of the protein CPS detection because low Istr intensities are deleterious to the p53CD–DNA complex stability at the electrode surface. These data highlight the utility of developing biosensors offering novel approaches for studying real-time macromolecular protein dynamics.

Enzymatic activity and catalytic hydrogen evolution in reduced and oxidized urease at mercury surfaces

It was originally shown [10] that urease retains its enzymatic activity when adsorbed at bare mercury and solid amalgam surfaces. However the opinion later prevailed that, when adsorbed at bare metal electrodes, proteins are irreversibly denatured. Here we confirm that urease is enzymatically active at a bare solid amalgam surface as found by Santhanam et al., and we show that this enzyme is equally active at a thiol-modified amalgam surface. We also show that it is the reduced form of urease, which is enzymatically active at Hg surfaces. Oxidation of the protein, resulting in formation of disulfide bonds, strongly decreases the enzyme activity. Using constant current chronopotentiometric stripping (CPS) we show that the exposure of surface-attached urease to negative potentials results in the protein unfolding. The extent of the unfolding depends upon the amount of time for which the protein is exposed to negative potentials, and at very short times this unfolding can be avoided. At thiol-modified Hg surfaces the protein is less vulnerable to the effects of the electric field. We conclude that the loss of enzymatic activity, resulting from a 10min exposure of the protein to −0.58V, is not due to reduction of the disulfide bonds as suggested by Santhanam et al. This loss is probably a result of protein reorientation, due to reduction of the Hg-S bonds (formed by accessible cysteines), followed by prolonged electric field effect on the surface-attached protein.

Electrochemical sensing of 2D condensation in amyloid peptides

The interfacial behavior of the model amyloid peptide octamer YYKLVFFC (peptide 1) and two other amyloid peptides YEVHHQKLVFF (peptide 2) and KKLVFFA (peptide 3) at the metal|aqueous solution interface was studied by voltammetric and constant current chronopotentiometric stripping (CPS). All three peptides are adsorbed in a wide potential range and exhibit different interfacial organizations depending on the electrode potential. At the least negative potentials, chemisorption of peptide 1 occurs through the formation of a metalsulfur bond. This bond is broken close to −0.6V. The peptide undergoes self-association at more negative potentials, leading to the formation of a “pit” characteristic of a 2D condensed film. Under the same conditions the other peptides do not produce such a pit. Formation of the 2D condensed layer in peptide 1 is supported by the time, potential and temperature dependences of the interfacial capacity and it is shown that presence of the 2D layer is reflected by the peptide CPS signals due to the catalytic hydrogen evolution. The ability of peptide 1 to form the potential-dependent 2D condensed layer has been reported neither for any other peptide nor for any protein molecule. This ability might be related to the well-known oligomerization and aggregation of Alzheimer amyloid peptides.

Electrocatalytic Monitoring of Metal Binding and Mutation-Induced Conformational Changes in p53 at Picomole Level

We developed an innovative electrochemical method for monitoring conformational transitions in proteins using constant current chronopotentiometric stripping (CPS) with dithiothreitol-modified mercury electrodes. The method was applied to study the effect of oncogenic mutations on the DNA-binding domain of the tumor suppressor p53. The CPS responses of wild-type and mutant p53 showed excellent correlation with structural and stability data and provided additional insights into the differential dynamic behavior of the proteins. Further, we were able to monitor the loss of an essential zinc ion resulting from mutation (R175H) or metal chelation. We envisage that our CPS method can be applied to the analysis of virtually any protein as a sensor for conformational transitions or ligand binding to complement conventional techniques, but with the added benefit that only relatively small amounts of protein are needed and instant results are obtained. This work may lay the foundation for the wide application of electrochemistry in protein science, including proteomics and biomedicine.

Protein Structure-Sensitive Electrocatalysis at Dithiothreitol-Modified Electrodes

Dithiothreitol (DTT)-mercury and DTT-solid amalgam electrodes are proposed for protein microanalysis by means of constant current chronopotentiometric stripping (CPS). At the DTT-modified hanging mercury drop electrode (DTT-HMDE), proteins at nanomolar concentrations produce the CPS peak H, which is due to the protein catalyzed hydrogen evolution. Self-assembled monolayers (SAMs) of DTT at the electrode surface protected surface-attached proteins from the electric field-driven denaturation, but did not interfere with the electrocatalysis. Using CPS peak H, native and denatured forms of bovine serum albumin (BSA) and of other proteins were easily distinguished. On the other hand, in usual slow scan voltammetry (scan rates between 50 mV/s and 1 V/s), the adsorbed BSA behaved as fully or partially denatured. BSA-modified DTT-HMDE was exposed to different potentials, E(B) for 60 s, followed by CPS measurement. Three E(B) regions were observed, in which either BSA remained native (A, -0.1 to -0.3 V), was denatured (B, -0.35 to -1.4 V), or underwent desorption (C, at potentials more negative than -1.4 V). At potentials more positive than the reduction potential of the DTT Hg-S bond (approximately -0.65 V against Ag|AgCl|3 M KCl), the densely packed DTT SAM was impermeable to [Ru(NH(3))(6)](3+). At more negative potentials, the DTT SAM was disturbed, but under conditions of CPS (with very fast potential changes), this SAM still protected the protein from surface-induced denaturation. Thiol-modified Hg electrodes in combination with CPS represent a new tool for protein analysis in biomedicine and proteomics.

Ex situ Voltammetry and Chronopotentiometry of Doxorubicin at a Pyrolytic Graphite Electrode: Redox and Catalytic Properties and Analytical Applications

AbstractEx situ (adsorptive transfer stripping) electrochemical techniques in connection with basal‐plane PGE have been applied to the study of redox and catalytic properties of doxorubicin (DOX). Cyclic and square‐wave voltammetry and constant current chronopotentiometric stripping (CPS) analysis were used to follow reversible reduction of DOX quinone moiety around −0.5 V and its coupling to catalytic oxygen reduction. CPS was for the first time used for sensitive ex situ determination of the DOX using the catalytic signal around −0.5 V in the presence of oxygen, allowing detection of femtomole amounts of DOX. We show that specific interaction of DOX with double‐stranded DNA can easily be monitored using the catalytic CPS signal.

From DC polarographic presodium wave of proteins to electrochemistry of biomacromolecules

History of electrochemistry of proteins and nucleic acids is briefly reviewed. The ability of proteins to catalyze hydrogen evolution at Hg electrodes was discovered almost 80 years ago in J. Heyrovský’s laboratory. This phenomenon was not sufficiently appreciated for several decades. Recently it has been shown that using constant current chronopotentiometric stripping (CPS) with hanging mercury drop, solid amalgam or Hg-film electrodes the CPS peak H is obtained with nanomolar concentrations of peptides and proteins. This peak is derived from the presodium wave but it has some new properties useful in protein research. It is sensitive to changes in protein structures and to protein redox states, representing a new tool for protein analysis applicable in biomedicine. Electroactivity of nucleic acids was discovered about 50 years ago. Electrochemistry of DNA and RNA is now a booming field because of its potential use in sensors for DNA hybridization and DNA damage. Quite recently it has been shown that electrochemistry can be applied also in polysaccharide analysis. A review with 99 references.