constant current chronopotentiometry Dotaz Zobrazit nápovědu
Metallothionein (MT) from rabbit liver was analyzed by differential pulse polarography, cyclic voltammetry, square wave voltammetry, and chronopotentiometric stripping analysis (CPSA) with a hanging mercury drop electrode under various conditions. The highest sensitivity of the MT determination was obtained with CPSA which produced a well-developed peak H due to catalytic hydrogen evolution at highly negative potentials. The highest peak H was obtained in borate buffer close to pH 8.0. In this medium, subnanomolar concentrations of MT were detectable. In the adsorptive transfer stripping (medium exchange) experiments, determination of few femtomoles of MT in 5-microL aliquots of the analyte was possible. CPSA determination of MT in various tissues of carp (Cyprinus carpio) yielded values in agreement with the published data.
- MeSH
- elektrody MeSH
- játra chemie MeSH
- králíci MeSH
- metalothionein analýza MeSH
- mikrochemie metody MeSH
- polarografie metody MeSH
- potenciometrie metody MeSH
- rtuť chemie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- metalothionein MeSH
- rtuť MeSH
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.
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.
- Klíčová slova
- Charged surface, Constant current chronopotentiometry, Electrode, Freezing, Scanning transmission electron microscopy, Serum albumin,
- MeSH
- adsorpce MeSH
- denaturace proteinů MeSH
- peptidy MeSH
- sérový albumin hovězí * chemie MeSH
- sérový albumin * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- peptidy MeSH
- sérový albumin hovězí * MeSH
- sérový albumin * MeSH
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.
- MeSH
- adsorpce MeSH
- elektrody * MeSH
- játra chemie MeSH
- katalýza MeSH
- králíci MeSH
- metalothionein chemie MeSH
- potenciometrie metody MeSH
- rtuť chemie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- metalothionein MeSH
- rtuť MeSH
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 I(str) 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.
- Klíčová slova
- Constant current chronopotentiometry, Deoxyribonucleic acid–protein binding, Electrochemical sensing, Mercury containing electrodes, Tumor suppressor protein p53,
- MeSH
- biosenzitivní techniky * MeSH
- DNA analýza chemie MeSH
- elektrochemické techniky * MeSH
- elektrody MeSH
- lidé MeSH
- nádorový supresorový protein p53 analýza chemie MeSH
- rtuť chemie MeSH
- sulfhydrylové sloučeniny chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
- nádorový supresorový protein p53 MeSH
- rtuť MeSH
- sulfhydrylové sloučeniny MeSH
Using constant current chronopotentiometry we showed that in 50 mM sodium phosphate (pH 7) bovine serum albumin and some other proteins were not significantly denatured at a bare mercury electrode while at higher phosphate concentrations they underwent electric field-driven denaturation on the electrode surface.
The capacitance measurement (dependence of the differential capacitance C of the electrode double layer on potential E, C-E curves), electrochemical impedance spectroscopy (frequency response of the impedance Z of the electrode double layer-EIS) and constant current chronopotentiometry (dependence of dt/dE on potential at constant current, chronopotentiometric stripping analysis-CPSA) have been used for electrochemical study of echinomycin and its interaction with single-stranded (ss) and double-stranded (ds) DNA at the hanging mercury drop electrode (HMDE). The capacitance measurement showed that echinomycin gives a pseudocapacitance redox peak strongly dependent on the a.c. voltage frequency at the potential of -0.53 V. This peak is observed with dsDNA-echinomycin complex as well, but not with ssDNA treated by echinomycin. Similar results were obtained using CPSA measurements. Thus capacitance measurements and CPSA can distinguish with the aid of the bis-intercalator echinomycin the single-stranded and double helical form of DNA adsorbed at the mercury electrode surface. Impedance measurement in connection with adsorptive transfer technique can find the differences between ssDNA and dsDNA, which promise to use this technique for detection of dsDNA in hybridisation reactions.
- Publikační typ
- časopisecké články MeSH
In contrast to previous reports claiming bovine serum albumin (BSA) denaturation at mercury surfaces, recently it has been shown that BSA and other proteins do not denature as a result of adsorption to the mercury electrodes at alkaline and neutral pH values. In this pH range, constant current chronopotentiometry (CPS) with mercury or solid amalgam electrodes can be used to distinguish between native, denatured and damaged BSA. Here we show that at acid pH values (around pH 4.5) native and denatured BSA yield almost the same CPS responses suggesting denaturation of native BSA at the electrode surface. Under these conditions BSA is, however, not denatured at the electrode at accumulation potentials (E(A) values) close to the potential of zero charge, but at E(A) values more negative than -0.8 V, after destabilization of the surface-attached BSA by electroreduction of some disulfide groups at about -0.48 V and by electric field effects at more negative potentials.
- MeSH
- adsorpce MeSH
- chlorid draselný chemie MeSH
- denaturace proteinů MeSH
- elektrody MeSH
- koncentrace vodíkových iontů MeSH
- potenciometrie MeSH
- povrchové vlastnosti MeSH
- pufry MeSH
- sérový albumin hovězí chemie MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chlorid draselný MeSH
- pufry MeSH
- sérový albumin hovězí MeSH
The hydrogen evolution reaction is catalyzed by peptides and proteins adsorbed on electrode materials with high overpotentials for this reaction, such as mercury. The catalytic response characteristics are known to be very sensitive to the composition and structure of the investigated biomolecule, opening the way to the implementation of a label-free, reagentless electroanalytical method in protein analysis. Herein, it is shown using the model peptide Cys-Ala-Ala-Ala-Ala-Ala that the interfacial organization significantly influences the catalytic behavior. This peptide forms at the electrode two distinct films, depending on the concentration and accumulation time. The low-coverage film, composed of flat-lying molecules (area per molecule of approximately 250-290 A(2)), yields a well-defined catalytic peak at potentials around -1.75 V. The high-coverage film, made of upright-oriented peptides (area per molecule of approximately 43 A(2)), is catalytically more active and the peak is observed at potentials less negative by approximately 0.4 V. The higher activity, evidenced by constant-current chronopotentiometry and cyclic voltammetry, is attributed to an increase in the acid dissociation constant of the amino acid residues as a result of the low permittivity of the interfacial region, as inferred from impedance measurements. An analogy is made to the known differences in acidic-basic behaviors of solvent-exposed and hydrophobic domains of proteins.
