Chronopotentiometric Stripping Research Articles

Interaction of lectin Sambucus nigra with sialylated trisaccharides in the presence of osmolytes. Chronopotentiometric sensing

Trisaccharides bind to their interaction partners-lectins relatively weakly, which makes detection of their complexes challenging. In this work, we show that an osmolyte presence improves the distinguishing complexes of lectin Sambucus nigra with trisialyllactoses with various binding affinities. The addition of osmolyte, non-binding sugar mannose significantly improved the precision of binding experiments performed using chronopotentiometric stripping at the electrode surface and fluorescence analysis in solution. Osmolytes minimized nonspecific interactions between binding sugar and lectin. Obtained findings can be utilized in any in vitro methods studying interactions of carbohydrates, respectively their conjugates with proteins. The study of carbohydrate interactions appears important since they play essential roles in a variety of biological processes including carcinogenesis.

Chronopotentiometric sensing of native, oligomeric, denatured and aggregated serum albumin at charged surfaces

In protein analysis, fast techniques applicable for preliminary tests of the protein structural changes are sought. We show that using constant current chronopotentiometric stripping peak H, small amounts of oligomeric, denatured and aggregated bovine serum albumin (BSA) can be easily distinguished from native form. Different behavior of native, denatured, and aggregated BSA could be explained by combination of their different adsorption at charged surface and accessibility of electroactive amino acid residues. Ability to discriminate between individual forms allows to use chronopotentiometric stripping for study of processes responsible for structural changes, such as freezing treatment.

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.

Electrochemistry of chitosan amino-glycan and BSA protein mixture under seawater conditions

Abstract We investigated the electrochemical properties of the models chitosan oligosaccharide (CHOS), an amino-glycan, and BSA protein, as major components of N-bearing polymeric organic material (N-POM) in seawater. The chronopotentiometric stripping method (CPS) and a hanging mercury drop working electrode (HMDE) were used to detect catalytic peaks (Ep around –1.8 V/–1.9 V vs. Ag/AgCl (3M KCl)) generated by the catalytic hydrogen evolution reaction (CHER). “Peak HOS” of CHOS was not observed under seawater conditions (pH 8.2). Since both analytes measured separately produced catalytic peaks in CPS after their adsorptive transfer (AdT) from seawater into slightly acidic and neutral buffers (pH-s 5.3–7.1), we investigated measurement conditions that might allow selective AdT detection of CHOS in the CHOS + BSA seawater mixture. Only one catalytic peak was observed in the AdT CPS of mixture, which was attributed to the involvement of both compounds in adsorption on HMDE and consequently in CHER. The height and Ep of this peak tended to correspond to the analyte with the higher concentration in the mixture. Regardless of the choice of pH or buffer, the combination of CPS measurement conditions or the rinsing mode by relevant solvents and solutions during AdT, BSA was not efficiently removed from the HMDE and selective detection of CHOS in the mixture was not achieved. These results will be applicable in the electrochemical analysis of amino-glycans in natural seawater samples containing a mixture of N-POM, but also represent an investigation of the basic electrochemical properties of CHOS and BSA.

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).

Electrodeposition of silver amalgam particles on screen-printed silver electrodes in voltammetric detection of 4-nitrophenol, bovine serum albumin and artificial nucleosides dTPT3 and d5SICS

Electrodeposition of silver amalgam particles (AgAPs) on various substrates represents a prospective approach to the development of a novel detection system applicable in the study of various electrochemically active substances, including nucleic acids and proteins. Herein, a double pulse chronoamperometric deposition of AgAPs on in-house fabricated screen-printed silver electrodes (SPAgE) has been optimized using the voltammetric signal of a model electrochemically reducible organic nitro-compound, 4-nitrophenol (4-NP). The surface morphology of SPAgE-AgAP was monitored by scanning electron microscopy with an energy dispersive X-ray spectrometer. A compact three-electrode sensor consisting of the working (substrate) SPAgE, counter graphite, and pseudo-reference Ag|AgCl electrodes was designed for analysis of electrochemically reducible compounds in a 96-well plate with about 150-μl sample volume per well. Herein optimized SPAgE-AgAP allowed detection of 4-NP down to 1 μmol l−1 using cyclic voltammetry. Advantageously, differential pulse voltammetry at SPAgE-AgAP allowed highly sensitive detection of artificial nucleosides dTPT3 and d5SICS (developed to expand genetic alphabet in semi-synthetic organisms) using their catalytic hydrogen evolution signals, with limits of detection of 0.4 pmol l−1. Moreover, these artificial nucleosides were easily detectable in the excess of natural nucleosides down to molar ratio 1:12000. Using constant current chronopotentiometric stripping and the catalytic peak H, we further demonstrated detection of a protein, bovine serum albumin, at the SPAgE-AgAP. Thus, we present the SPAgE-AgAP as a potent tool applicable in simple, fast, and sensitive electrochemical detection of reducible or catalytically active species, with prospective applications in hot research areas including chemical and synthetic biology.

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.

Background Concentration of Copper and Cadmium in Water from a High Industrialized and Urban Coastal System (Baixada Santista, Brazil)—Contribution to a Monitoring Program

The Baixada Santista coastal region is well known in the context of the high atmospheric, soil and aquatic pollution levels derived from port, industrial, domestic and urban activities existent there, mainly in function of the Cubatão industrial pole and port of Santos. The contamination by trace metals in this region is rarely measured in water column when compared with metal determinations in sediment studies. This study aims to evidence the levels of dissolved copper and total cadmium concentrations in estuarine and seawater waters using electrochemical analysis as a chronopotentiometric stripping polarography, which could improve the environmental monitoring program. The study was performed in the Bay of Santos reaching two estuarine channels (Santos and São Vicente Channels) in two tide periods in summer 2001. This region is influenced by terrestrial and anthropogenic inputs of trace metals to seawater. The results showed that dissolved copper and total cadmium in water presented the maxima concentrations of 45.4 nM and 2.6 nM respectively. They not reached the limits proposed by Brazilian Environmental Law (CONAMA), that indicate, as reference, a maximum of 123 nM and 350 nM for dissolved copper and total cadmium, respectively and, considering seawater coastal system (salinity > 30, class 2) and also brackish water (class 2). Although the values were under the maximum recommended for each metal, the concentrations were not negligible in relation to the other polluted areas in the world. The values distribution along the system showed a progressive increase in direction to the inner part of the system that naturally compromises the local biota whose is more exposed to the bioaccumulation processes, and this data corresponds to an important reference preterit value to the monitoring program. The trace metal bioaccumulation by mangrove vegetation and by the biota in the internal part of the estuarine channels increase the risk of the poorly population that living in the internal part of the estuary, used to the seafood consumption. The contribution of this study is important to guide the protective policies destined to recuperate the natural conditions of this system, mainly considering that in the decade 1980, there was a case of toxic contamination, causing hydrocephaly in newborns. It proves the existence of risks to the human health and to the balance of the ecosystem, even with the decrease of pollution after that, the environmental evaluation needs to know the background values to guide environmental protection.

Bovine Serum Albumin Catalysed Hydrogen and Deuterium Evolution at Mercury Electrodes.

The hydrogen evolution reaction (HER), catalysed by proteins at mercury electrodes and reflected in chronopotentiometric stripping peak H, provides a label-free and reagentless analytical technique that is sensitive to protein structure. Here we show how the kinetic isotope effect affected the HER catalysed by the protein bovine serum albumin (BSA). We found that the deuteron bond, which is stronger than that of a proton, contributed to less effective transport of deuterons mediated by BSA at the Hg|D2 O interface, and enhanced structural stability of the surface-attached native BSA in D2 O solution. A structural transition was also observed in the surface-attached urea-denatured BSA, and is probably due to the destabilisation of some secondary structural remnants retained by the 17 SS-bonds. Because the catalytically active groups involved in proton or deuteron transfer in native proteins are often exposed towards solutions and their protons exchange almost instantly, no signs of H/D exchange were observed in native BSA using peak H under the given conditions.

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.

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.

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.

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.

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.

Catalytic Deuterium Evolution and H/D Exchange in DNA

AbstractRecently, it has been shown that DNA catalyzes the hydrogen evolution reaction (HER) at mercury‐containing electrodes, which provide voltammetric and chronopotentiometric stripping (CPS) peaks , allowing label‐free DNA determination at very low concentrations. Here, we compared DNA catalytic HER in H2O with catalytic deuterium evolution reaction (DER) in D2O. We show that in D2O single‐stranded (ss) and double‐stranded (ds) DNAs produced CPS peaks due to DER. Compared to peaks obtained in H2O, peaks were smaller and appeared at more negative potentials. Under conditions where dsDNA did not produce any peak in D2O, this DNA produced in H2O peak . These results suggested a lack of accessible bases in the D2O surface‐attached dsDNA. This might be attributed to the greater stability of deuterated dsDNA, better resisting the electric field effects at the negatively charged interface. The process of protonation of deuterated ssDNA was very fast in contrast to that of dsDNA, which was much slower and could be followed by CPS in a time range of tens of minutes.

Structural Stability of Peptidic His-Containing Proton Wire in Solution and in the Adsorbed State.

Molecular wires are functional molecules applicable in the field of transfer processes in technological and biochemical applications. Besides molecular wires with the ability to transfer electrons, research is currently focused on molecular wires with high proton affinity and proton transfer ability. Recently, proposed peptidic proton wires (H wires) are one example. Their ability to mediate the transport of protons from aqueous solutions onto the surface of a Hg electrode in a catalytic hydrogen evolution reaction was investigated by constant-current chronopotentiometric stripping. However, elucidating the structure of H wires and rationalizing their stability are key requirements for their further research and application. In this article, we focus on the His (H) and Ala (A)-containing peptidic H wire A3-(H-A2)6 in solution and after its immobilization onto the electrode surface in the presence of the secondary structure stabilizer 2,2,2-trifluoroethanol (TFE). We found that the solvent containing more than 25% of TFE stabilizes the helical structure of A3-(H-A2)6 not only in solution but also in the adsorbed state. The TFE efficacy to stabilize α-helical structure was confirmed using high-resolution nuclear magnetic resonance, circular dichroism, and molecular dynamics simulation. Experimental and theoretical results indicated A3-(H-A2)6 to be a high proton-affinity peptidic H wire with an α-helical structure stabilized by TFE, which was confirmed in a comparative study with hexahistidine as an example of a peptide with a definitely disordered and random coil structure. The results presented here could be used for further investigation of the peptidic H wires and for the application of electrochemical methods in the research of proton transfer phenomena in general.

J. Heyrovský's Oscillographic Polarography. Roots of Present Chronopotentiometric Analysis of Biomacromolecules

AbstractIn 1941 J. Heyrovský invented Oscillographic Polarography (OP) with controlled alternating current (a.c.). The method was developed during the World War and the first commercially available instruments for OP were produced in Czechoslovakia at the beginning of the 1950’s. Such instruments offered derivative and cyclic OP curves allowing simple analysis of various compounds and stimulated the publication of hundreds of papers. Thanks to OP, electrochemical analysis of DNA started already in 1958. According to the present nomenclature, OP can be denominated as a.c. chronopotentiometry. Surprisingly, authors who later described derivative and cyclic chronopotentiometry did not consider J. Heyrovský’s OP methodology in their work. Present electrochemical studies of biomacromolecules take advantage of modern instruments for constant current chronopotentiometric stripping, allowing DNA and protein structure‐sensitive analysis. Recent progress in these studies is reviewed.