【作者】 彭勇；

【导师】 刘又年；

【作者基本信息】 中南大学 ， 应用化学， 2011， 博士

【摘要】 电化学方法具有快速、灵敏和操作简单等优点,由于大多数多肽或蛋白质不具有电化学活性,阻碍了电化学方法在生物化学领域的应用。通过电活性标记物的引入,可使本身不具有电活性或在电极表面电子传递速率较慢的多肽或蛋白具有电化学活性。在生命过程中的许多生理现象与电流或电势的变化密切相关。因此,研究生物组分的氧化还原特性对探讨疾病的致病机理、研发新型药物等具有重要的意义。基于上述考虑,本论文的主要内容有：(1)以二茂铁甲酸为起始原料,采用液相合成法得到具有电活性的还原型谷胱甘肽-二茂铁(GSH-Fc),总收率为12.5%。利用电化学方法研究了GSH-Fc与BSA的相互作用,结合比和结合常数分别为1.41±0.06和6.53±2.01×106 L-mol-1,结合位点位于BSA的subdomain IIA。利用荧光光谱方法验证了电化学方法的可靠性,同时荧光方法表明GSH-Fc与BSA结合以疏水作用和氢键为主。利用电化学方法研究了GSH-Fc与金属硫蛋白(MT)的相互作用。实验结果表明GSH-Fc在Zn7-MT修饰金电极上有一对很好的氧化还原峰,Epa=0.218V, Epc=0.154V,ΔAEp=64mV,Ipa/Ipc=1.03。而GSH-Fc在裸金电极上的氧化还原电位分别为Epa’=0.205 V和Epc’=0.147 V,ΔEp’=58mV,Ipa’/Ipc’=1.02,说明MT与GSH-Fc之间具有特殊相互作用,结合比和结合常数分别为1.78±0.15和5.54±0.43×108L-mol-1,在GSH-Fc或GSH)和GSSG共存的溶液中,Zn(Ⅱ)可以从MT分子中释放。(2)构建了11-二茂铁基十一烷基-1-硫醇(FcC11SH)和聚丙烯酰胺包覆的过氧化氢酶(HRP)混双分子膜修饰电极实现过氧化氢(H2O2)的微量检测。通过调整组装HRP的硫醇链长以及FcC11SH介体,电子能在FcC11SH和HRP之间转移。该电极安培检测H2O2最低浓度为0.64nmol-L-1,检测动态范围宽。HRP的包覆没有影响电子的传递以及H2O2的扩散,更重要的是通过与未包覆的酶电极、商业化的无线HRP/polymer电极对比,包覆后的酶能有效的提高酶的稳定性,在3星期后,酶的活性能保持90%,且能有效的防止介体的渗漏。此外,FcC11SH/包覆的HRP电极能快速测定样品中H2O2的浓度,具有较好的重现性。采用FcC11SH和葡萄糖氧化酶(GOD)混双分子膜电极研究了第二代酶传感器,由于FcC11SH电位比较高,因此在实际样品检测时,许多电活性物质如抗坏血酸(AA)、尿酸(UA)和对乙酰氨基酚(ACP)容易氧化,对结果造成干扰。利用网状玻璃碳(RVC)材料的高表面积以及高电流密度设计一种全新的葡萄糖传感器检测装置。通过联用FcC11SH和GOD混双分子膜组装的薄层电极,葡萄糖的浓度在0.05～40 mmol·L-1之间呈现良好的线性关系,检测限为3.6μmol-L-1。通过调整流动注射的流速和RVC电极的电位,具有电活性物质基本上完全被氧化,且氧化后的产物对酶电极不会产生干扰信号。应用该装置实现了实际样品的实时在线检测。(3)阿尔茨海默氏病(AD)是一种神经退行性疾病,主要病因是病人大脑中β-淀粉样肽(amyloidβ-peptide,Aβ)的异常聚集,因此抑制Aβ的异常聚集能有效的延缓或治疗老年痴呆症。大量研究表明Lys-Leu-Val-Phe-Phe(KLVFF)五肽序列能够与Aβ结合从而阻止Aβ的聚集。但KLVFF水溶性及脂溶性差,易被酶解。因此,采用固相合成法合成了二茂铁标记的水溶性较好的阻断肽Fc-KLVFFK6,该阻断肽含有能与Aβ识别并阻断Aβ形成纤维的疏水性片段。利用二茂铁的电化学活性研究了该阻断肽对Aβ聚集的动力学,表明Fc-KLVFFK6能较好的阻断Aβ纤维的聚集,同时利用荧光光谱和原子力显微镜研究了Fc-KLVFFK6和KLVFFK6抑制Aβ纤维的聚集,结果和电化学方法一致。(4)帕金森病(PD)是第二大最常见的神经退行性疾病,主要特征是多巴胺能神经元细胞的丢失引起多巴胺的缺乏,以及金属离子(Cu和Fe)沉积,目前致病机理尚未清楚。α-syn在PD的神经病理中具有重要的作用,能与Cu(Ⅱ), Zn(Ⅱ)和Fe(Ⅱ)结合,因此本研究利用质谱分析了α-syn与Fe(Ⅱ)相互作用,证明α-syn与Fe(Ⅱ)相互作用能形成1：1的配合物,该配合物的氧化电位和还原电位分别为0.143V和-0.093V,根据能斯特方程,α-syn-Fe(Ⅲ)的平衡常数KA为1.2×1013 L·mol-1。由于Fe(Ⅲ)溶度积常数Ksp为1.0×10-38,在生理条件下α-syn-Fe(Ⅲ)不稳定,容易水解,且α-syn与Fe(Ⅱ)结合后,在空气中能与O2反应生成H2O2。通过荧光光谱方法验证了H2O2生成,并探讨了α-syn与Fe(Ⅱ)结合后在PD中的诱导的氧化应激步骤、a-syn的聚集以及在细胞内Fe(Ⅱ)的传递。Cu(Ⅱ)能加速a-syn的聚集形成不同神经毒性的聚集,然而Cu(Ⅱ)是具有氧化还原活性,而Cu(Ⅱ)和许多蛋白或多肽形成的配合物与神经退行性疾病的氧化步骤相关。因此,在本研究中,合成了a-syn的两个具有与Cu(Ⅱ)结合较强的片段：α-syn(1-19)和α-syn(20-50),利用质谱分析了α-syn,α-syn(1-19)和α-syn(20-50)与Cu(Ⅱ)相互作用,表明α-Syn及其片段多肽能和Cu(Ⅱ)形成配合物,并利用电化学方法研究了其电化学行为,其标准氧化还原电位分别为0.04V,0.072V和0.025V,在O2存在的条件下能催化O2的还原,生成H2O2,且抗坏血酸(AA)和多巴胺(DA)溶液分别加入α-Syn和Cu(Ⅱ)的混合溶液,与PD病直接相关的DA电化学行为没有发生改变,而AA能还原α-Syn蛋白和多肽与Cu(Ⅱ)的配合物,由此可知α-Syn和Cu(Ⅱ)的配合物在AA或者其他具有氧化还原活性片的物质(如GSH和NADH)能导致氧化应激步骤,从而可能导致多巴胺细胞的损伤。更多还原

【Abstract】 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 (ΔEp=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 (ΔEp’=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(Ⅱ)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 3.6μmol·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 (Aβ), so inhibition of the Aβaggregation 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 Aβ, could interact with the corresponding residues of Aβvia self-cognition and disrupted the self-assembly of Aβinto fibrils. We used electrochemical method to investigate the interaction and the kinetic process of inhibitor with Aβin vitro. The electrochemical results revealed Fc-KLVFFK6 inhibits Aβaggregation well. The inhibitory effect on Aβwas 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.α-Synuclein (α-syn), a presynaptic protein believed to play an important role in neuropathology in Parkinson’s disease (PD), is known to bind Cu(Ⅱ), Zn(Ⅱ) and Fe(Ⅱ). We used the mass spectrum to investigate the interaction of a-syn with Fe(Ⅱ), 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(Ⅲ) was 1.O×10-38, a-syn-Fe(Ⅲ) was unstable in the physiologic condition and had the stronger tendency to hydrolyze. When a-syn binded with Fe(Ⅱ), the complex can react with O2 to produce H2O2 which was proved by fluorescence kit. We also studied the process of a-syn-Fe(Ⅱ) induced the oxidative stress, a-syn aggregation and transporting Fe(Ⅱ) in cells of PD. Cu(Ⅱ) 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(Ⅱ) 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(Ⅱ), a-syn(1-19)-Cu(Ⅱ) and a-syn(20-50)-Cu(Ⅱ) 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.更多还原