Research On Electrochemistry Based On The Biological Activity Of Peptides And Proteins

The interaction between peptide and protein has been investigated extensively using electrochemical method. However, most of peptides or proteins are electro-inactive which limits the application of electrochemical method. Introducing of an electroactive group should allow bimolecular that are either electro-inactive or do not exhibit reversible voltammetric responses to be analyzed and detected. The metabolism and various physiological phenomena in human and animal were almost related to the current or potential change; meanwhile, it is important to investigate the redox mechanism of some biological components for searching the pathogenesis of the diseases and developing new drugs. In the thesis, the following studies were carried out.(1) Glutathione (GSH) tagged with a ferrocene (Fc) label at its C-terminal was synthesized via coupling ferrocene monocarboxylic to glutathione in liquid-phase with the yields of 12.5%. The interaction of GSH-Fc with bovine serum albumin (BSA) was investigated, and a binding ratio of 1.41±0.06 (GSH-Fc/BSA) and an affinity constant Ka of 6.53±2.01 X 106 L·mol-1 were determined. And the voltammetric study in solution or surface confirmed that GSH-Fc binds at subdomain IIA of BSA with high affinity. These results compare well with those measured by fluorescence, the hydrophobic association and hydrogen bonding played the important roles in the binding process of GSH-Fc with BSA through fluorescence method. Electrochemical method was also employed to study the interaction between GSH-Fc and Zn7-MT. A pair of well-defined voltammetric peaks with the anodic peak potential (Epa=0.218 V) and cathodic peak potential (Epc=0.154 V) were observed for GSH-Fc at Zn7-MT modified electrode (?Ep=77 mV,Ipa/Ipc=1.13). However, the anodic and cathodic peak potentials of 0.205 V and 0.147 V, respectively, were observed for GSH-Fc at bare gold electrode (?Ep'=58 mV,Ipa'/Ipc'=1.02). It indicates that GSH-Fc undergoes a reversible electron transfer reaction and a specific interaction between GSH-Fc and MT, the binding constant and the binding ratio were 5.54±0.43×108 L·mol-1 and 1.78±0.15, respectively. Furthermore, Zn(?)can be released from MT in the present of GSH-Fc(or GSH) and GSSG.(2) We constructed a mixed monolayer comprising ferrocenylalkanethiol and encapsulated horse radish peroxidase (HRP) at a gold electrode for enhanced amperometric detection of H2O2 at trace levels. By tuning the alkanethiol chain lengths that tether the HRP enzyme and the ferrocenylalkanethiol (FcC11SH) mediator, facile electron transfer between FcC11SH and HRP can be achieved. The electrocatalytic reaction proceeding at the mixed monolayer-modified electrode was used to attain a low amperometric detection level (0.64 nmol-L-1) and a dynamic range spanning over three orders of magnitude. Not only does the thin hydrophilic porous HRP capsule allow facile electron transfer, it also enables H2O2 to permeate. More significantly, the enzymatic activity of the encapsulated HRP is able to retain for a considerably longer period (more than three weeks) than naked HRP molecules attached to an electrode or those wired HRP/polymer electrode. Furthermore, the mixed monolayer-modified electrode is capable of rapidly and reproducibly detecting H2O2 present in complex sample media. The mixed monolayer of ferrocenylalkanethiol (FcC11SH) and glucose oxidize (GOD) electrodes were also employed to investigate the second generation enzyme sensor. As for the high potential of the FcC11SH oxidation, many electroactive interferences in samples such as the ascorbic acid (AA), uric acid (UA) and acetaminophen (ACP) can be oxidized, resulting in the errors. So we utilized the large surface area and high current densities reticulated vitreous carbon (RVC) electrode combined with thin-layer electrode assembled with FcC11SH/GOD to design a novel glucose sensor system. It had a good linear of the catalytic current with the glucose concentration (0.05?40 mmol·L-1), the detection limit was 3.6?mol·L-1. Through adjusting the flow rate and the applied potential at RVC, the electroactive interferences were almost oxidized and did not interfere the enzyme electrode.(3) Alzheimer's disease (AD) is a devastating degenerative disorder and the main etiology of AD is the deposition of aggregates composed by a misfolded form of the amyloid beta peptide (A?), so inhibition of the A?aggregation can effective delay or cure. It was proved that Lys-Leu-Val-Phe-Phe (KLVFF) can inhibit the AP aggregation. However, the lipophilicity, water solubility and proteolytic stability of KLVFF were poor. Herein, we employed the ferrocene moiety as an electrochemical marker to label the hendecapeptide KLVFFK6, designed and synthesized a novel N-ferrocenoyl peptide Fc-Lys-Leu-Val-Phe-Phe-Lys-Lys-Lys-Lys-Lys-Lys (Fc-KLVFFK6) for an inhibitor, which contained the hydrophobic core of A?, could interact with the corresponding residues of A?via self-cognition and disrupted the self-assembly of A?into fibrils. We used electrochemical method to investigate the interaction and the kinetic process of inhibitor with A?in vitro. The electrochemical results revealed Fc-KLVFFK6 inhibits A?aggregation well. The inhibitory effect on A?was also investigated using thioflavin T (Th-T) fluorescence probe and AFM.(4) Parkinson's disease (PD) is the second most common neurodegenerative disease, and it is characterized by a progressive loss of the dopaminergic cells in the substantial nigra which is a small brain region producing dopamine and high concentrations of the metal ions(Cu and Fe), but the pathogenic mechanism of PD is unclear at present.?-Synuclein (?-syn), a presynaptic protein believed to play an important role in neuropathology in Parkinson's disease (PD), is known to bind Cu(?), Zn(?) and Fe(?). We used the mass spectrum to investigate the interaction of a-syn with Fe(?), and the results revealed they could form a complex and the binding ration was unity. Also the oxidized and reduced potential of complex was 0.143 V and-0.093 V, respectively. The value of equilibrium constant (KA) was 1.2×1013 L-mol-1. As for the Ksp of Fe(?) was 1.O×10-38, a-syn-Fe(?) was unstable in the physiologic condition and had the stronger tendency to hydrolyze. When a-syn binded with Fe(?), the complex can react with O2 to produce H2O2 which was proved by fluorescence kit. We also studied the process of a-syn-Fe(?) induced the oxidative stress, a-syn aggregation and transporting Fe(?) in cells of PD. Cu(?) has been shown to accelerate the aggregation of a-syn to form various toxic aggregates in vitro. However, copper is a redox active metal and its complexes with other amyloidogenic proteins/peptides have been linked to oxidative stress in major neurodegenerative diseases. So we synthesized the two a-syn N-terminus peptides (a-syn(1-19) and a-syn(20-50)) which they can form complexes with Cu(?) and the results were confirmed by electrospray-mass spectrometry. We utilized the electrochemical methods to study the electrochemical behaviors of these complexes, and the redox potential of a-syn-Cu(?), a-syn(1-19)-Cu(?) and a-syn(20-50)-Cu(?) was 0.04 V,0.072 V and 0.025 V, respectively. These complexes can catalyze O2 to reduce to H2O2 when bubbled with O2 to solution. While easily oxidized cellular species such as ascorbic acid can undergo electron transfer reaction with the copper complex of a-syn, dopamine, the most important neurotransmitter relevant to PD, cannot be directly oxidized, while AA can reduce the copper complex of a-syn. Herein, the copper complex of a-syn can react with AA or redox molecules (e.g., GSH and NADH) to induce the oxidative stress and could lead the dopaminergic cell damage.