Native and denatured forms of proteins can be discriminated at edge plane carbon electrodes.
ABSTRACT In an attempt to develop a label-free electrochemical method for detection of changes in protein structures based on oxidizability of tyrosine and tryptophan residues we tested different types of carbon electrodes. We found that using edge plane pyrolytic graphite electrode (EPGE) we can discriminate between native and denatured forms of human serum albumin (HSA) and of other proteins, such as bovine and chicken serum albumin, aldolase and concanavalin. Treatment of natively unfolded α-synuclein with 8 M urea resulted only in a small change in the tyrosine oxidation peak, in a good agreement with absence of highly ordered structure in this protein. Using square wave voltammetry with EPGE we were able to follow the course of HSA denaturation at different urea concentrations. The electrochemical denaturation curve agreed reasonably well with that based on intrinsic fluorescence of tyrosine and tryptophan. It can be expected that the electrochemical method will be applicable to a large number of proteins and may become useful in biomedicine and proteomics.
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ABSTRACT: Several severe neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and prion-associated transmissible spongiform encephalopathies, have been linked to dysregulation of specific proteins capable of self-assembly into deleterious fibrillar aggregates termed amyloids. A wide range of analytical techniques has been used to clarify the mechanisms of these protein-misfolding processes, in the hope of developing effective therapeutic treatment. Most of these studies have relied heavily on conventional methods of protein characterization, notably circular dichroism spectroscopy, thioflavin T fluorescence, transmission electron microscopy, and atomic force microscopy, which are particularly suitable for monitoring later-stage aggregate formation. Although electrochemical methods of protein detection have existed for some time, they have only recently gained prominence as a powerful tool for studying the early stages of protein aggregation during which the more toxic soluble amyloid species form. Electrochemical detection methods include direct detection of intrinsic redox-active amino acid residues, protein-catalyzed hydrogen evolution, use of extrinsic β-sheet binding mediators, and impedance spectroscopy. In this review, we evaluate the use of electrochemistry for study of protein aggregation related to neurodegenerative disorders.Analytical and Bioanalytical Chemistry 03/2013; · 3.66 Impact Factor
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ABSTRACT: 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.Analytica Chimica Acta. 01/2014;
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ABSTRACT: A novel aptamer and surface plasmon resonance (SPR)-based sensor was developed for the label-free detection of lysozyme. The aptasensor is characterised by a detection limit of 1 μg mL(-1) and a linear range of 5-50 μg mL(-1). As an application, we examined the usefulness of the aptasensor for monitoring the early stages of the aggregation of lysozyme. It was surprisingly found that, despite a significant decrease in monomer content during aggregation, the response of the aptasensor for protein solutions aged for 12 hours was similar to that for the fresh protein. To correlate the results obtained with the aptasensor with the composition of lysozyme solutions at various time points, we examined them in detail by atomic force microscopy (AFM), thioflavin T fluorescence, size-exclusion chromatography (SEC) and Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF-MS). All methods together indicated that during the initial hours of aggregation, the protein solutions contained small lysozyme oligomers (mainly dimers) and decreasing amounts of monomers. Our results thus suggest that the aptamer also recognizes lysozyme dimers/oligomers. A higher non-specific binding was observed for the aggregated lysozyme at the surface of the aptasensor as compared to the native protein. This was attributed to the hydrophobic patches which are exposed by the unfolded lysozyme and/or oligomer species, allowing for different adsorption and organisation at the surface of the aptasensor. This hypothesis is supported by square wave voltammetry (SWV) studies using solutions of aggregated lysozyme. A higher electrochemical signal due to the direct oxidation of tyrosine/tryptophan residues was observed for aged protein solutions as compared to the fresh solution, indicative of an increased number of such exposed electroactive residues and of overall increased surface hydrophobicity of the protein. Our work presents a label-free lysozyme aptasensor that is useful not only for the detection of the protein monomer but also for observing the onset of aggregation. The approach can be extended to other proteins which are prone to aggregation.The Analyst 05/2013; · 4.23 Impact Factor