Sensitive Electrochemical Detection of Native and Aggregated α‐Synuclein Protein Involved in Parkinson's Disease

Abstract

The aggregation of a-synuclein, a 14 kDa protein, is involved in several human neurodegenerative disorders, including Parkinson×s disease. We studied native and in vitro aggregated a-synuclein by circular dichroism (CD), atomic force microscopy (AFM) and electrochemical methods. We used constant current chronopotentiometric stripping analysis (CPSA) to measure hydrogen evolution catalyzed by a-synuclein (peak H) at hanging mercury drop electrodes (HMDE) and square-wave stripping voltammetry (SWSV) to monitor tyrosine oxidation at carbon paste electrodes (CPE). To decrease the volume of the analyte, most of the electrochemical measurements were performed by adsorptive transfer (medium exchange) from 3 ± 6 mL drops of a-synuclein samples. With both CPE and HMDE we observed changes in electrochemical responses of a-synuclein corresponding to protein fibrillization detectable by CD, fluorescence and AFM. Aggregation-induced changes in peak H at HMDE were relatively large in strongly aggregated samples, suggesting that this electrochemical signal may find use in the analysis of early stages of asynuclein aggregation. This assumption was documented by marked changes in the peak H potential and height in samples withdrawn at the end of the lag and the beginning of the elongation phase. Native a-synuclein can be detected down to subnanomolar concentrations by CPSA.

Electroactivity of Nonconjugated Proteins and Peptides. Towards Electroanalysis of All Proteins

“…In addition to BSA we observed large peaks of urea-denatured human serum albumin, g-globulin, myoglobin and a-crystalline as compared to much smaller peaks of these proteins in their native states [123]. On the other hand such difference was not found in natively unfolded proteins, such as a-synuclein [90,123] and p53 C-terminal peptides (Ostatna and Palecek, unpublished results). Sensitivity of determination of ureadenatured BSA was very high; even 2 nM BSA yielded a well-developed peak H, while 2 nM native BSA did not produce any signal [123].…”

“…Trp is not contained but there are 4 Tyr in AS, three of them located in the C-terminal region (comprising aas 96 -140), which region is presumably disordered under most conditions [202]. We studied the AS aggregation in vitro using SWV to measure oxidation of Tyr residues at carbon electrodes and CPS peak H at HMDE [90] in addition to the fluorescence of thioflavin T and circular dichroism known to reflect fibril formation. We showed a decrease of peak H by about 40% accompanied by shift of E p by > 30 mV to less negative values in early stages of the AS incubation, when optical methods indicated no changes [90].…”

Constant Current Chronopotentiometry and Voltammetry of Native and Denatured Serum Albumin at Mercury and Carbon Electrodes

“…We denominated it as peak H because of J. Heyrovský, Hydrogen evolution and High sensitivity [1,29]. In recent years we have been uncovering further interesting properties of this peak and particularly its surprising sensitivity for global and local changes in protein conformation [6,10,11,18,32,33]. We found conditions under which peak H is capable to discriminate between reduced and oxidized peptides [18] and proteins [25], native and aggregated proteins [17,18] and to detect conformational changes resulting from a single amino acid exchange in mutant proteins [6] as well as global changes observed in denatured proteins.…”

Chronopotentiometric Stripping Analysis of Gelatinase B, Collagen and Their Interaction

“…Disadvantages include high standard deviations and time of analysis at high stripping currents [9]. CPSA has been used for the detection of several biologically important peptides [10,11] and proteins such as metallothionein [12 -18], a-synuclein protein [19], MutS protein [20], glutathione-S-transferase [21], thrombin [22]. Moreover, Ostatna et al showed this electrochemical method can be employed to study structural changes of bovine serum albumin [23,24].…”