金属蛋白MT的电化学行为以及金属蛋白p53与DNA相互作用的研究

【作者】 李雪；

【导师】 王建秀；

【作者基本信息】 中南大学 ， 分析化学， 2010， 硕士

【摘要】 金属蛋白是由氨基酸和金属离子结合而成的。金属离子作为金属蛋白中重要的辅因子,承担着活性中心或结构中心的作用。金属硫蛋白与癌症抑制转录因子p53是两种重要的金属蛋白。金属硫蛋白(Metallothionein, MT)是一类低分子量,高半胱氨酸含量,能结合大量重金属离子的蛋白质。MT广泛存在于人体、动物、植物以及微生物体内,它参与必需金属的平衡代谢和重金属解毒过程。MT电化学行为的研究对阐述氧化还原诱导的MT电子转移与金属释放过程以及环境毒理学方面的作用具有重要意义。到目前为止,MT电化学行为的研究几乎都是在汞电极上进行的,由于MT与汞之间较强的结合力,MT中的Cd和Zn离子有可能被汞所取代。此外,汞电极的机械强度差且容易产生污染。引入具有选择性渗透作用的Nafion膜,有望克服上述缺陷。本文采用新型的Nafion-汞膜修饰电极(NCMFEs)对两种形态的MT,即Zn7-MT和CdT-MT进行了定量研究。分别在-1.141和-0.774 V处观察到Zn7-MT和Cd7-MT的氧化还原峰。结合电化学和原子吸收计算出电极表面每个MT分子中具有电活性的金属-巯基(M-Cysteine, M=Zn,Cd)侧链的数量分别为2.03和0.62。表明在一个MT分子中,并不是所有金属-半胱氨酸侧链都参与氧化还原反应,只有距离电极表面较近的基团才能够参与电子转移过程。上述工作对于阐述MT在环境毒理学和细胞内金属转移过程的机制具有重要的作用。肿瘤抑制转录因子p53在遏止肿瘤细胞生长、DNA修复、以及细胞程序化凋亡等方面扮演着十分重要的角色,因而有“基因组卫士”的美称。p53是一种结合Zn离子的蛋白质,其生物学功能和与DNA键合的特性有关。p53蛋白质中与DNA键合的区域含有一个锌指结构,锌指结构中锌离子对于维持野生型p53(没有发生突变的型体)的构型、稳定性及其与DNA的键合活性至关重要。目前常见的检测p53与DNA相互作用的方法有凝胶迁移滞后法、酶联免疫吸附分析法(ELISA)、以及电化学等。与上述方法相比,荧光分析法是一种简单、有效且灵敏的检测手段。通过引入能嵌入DNA螺旋中的荧光染料探针,检测了p53与DNA相互作用。另外,通过荧光共振能量转移(FRET),其中p53作为供体,DMACA修饰的DNA作为受体,来指示p53与DNA之间的相互作用。上述方法对灵敏、简便地检测p53与DNA的相互作用过程提供了重要依据。更多还原

【Abstract】 Metalloproteins are composed of amino acids and metal ions.As the most important cofactors of proteins, metal ions are responsible for the active or structural sites in metalloproteins.Metallothionein and p53,the transcription factor protein, are two kinds of the most important metallproteins.Metallothionein (MT) is a type of metalloproteins characterized by a large number of cysteine residues, low molecular weight and the absence of aromatic or histidine residues.It exerts its functions in regulating essential metals, detoxifying heavy metals, scavenging free radicals and controlling the intracellular redox potential.Investigation of electrochemistry of MT is of great significance in illustrating the role of MTs in environmental toxicology and in intracellular metal transfer. Up to now, characterization of the redox properties of MT has been carried out at mercury electrodes.However, the use of mercury could result in replacement of Cd2+ or Zn2+ inherent in MTs by Hg2+ due to the higher binding affinity of MT toword Hg. Furthermore, mercury electrodes are known to be mechanically unstable and prone to surface contamination. To alleviate these problems, voltammetric studies of Zn7-MT and Cd7-MT have been carried out at Nafion-coated mercury film electrodes (NCMFEs).Two well-defined redox waves at-0.774 and -1.141 V, which correspond to the reduction reactions of Cd7-MT and Zn7-MT complexes, respectively, were observed. Via electrochemistry and flame atomic absorption spectrophotometry (AAS), the amount of the redox-active metal-cysteine side chains per MT molecule entrapped into the NCMFEs was determined to be 2.03 and 0.62 for Zn7-MT and Cd7-MT, respectively. The above results indicate that not all of the metal-cysteine side chains of MTs entrapped into the NCMFEs have participated in the interfacial electron transfer (ET) reactions, only those that are positioned in close vicinity of the electrode with favorable orientation are accessible for facile ET reactions.Voltammetric characterization of MTs at Nafion-coated mercury film electrodes is of great significance in illustrating the role of MTs in environmental toxicology and in intracellular metal transfer.The tumor suppressor p53,also referred to as the "guardian of the genome",plays important roles in maintenance of genome integrity, DNA repair and programmed cell death. p53 is a Zn-containing protein with biological functions depending on its DNA-binding properties. p53 is characterized with a zinc-finger-like motif in which one zinc ion is tetrahedrally coordinated to three cysteins and one histidine. The zinc ions are requisite for maintenance of wild-type p53 conformation, stability and its sequencespecific DNA-binding activity. Common methods for the determination of the interaction between p53 and DNA include enzyme-linked immunosorbant assay (ELISA), electrophoretic mobility shift assay, and electrochemistry. In comparison with the above-metioned techniques, the fluorescence spectrometry is simple, sensitive, and inexpensive to implement. The interaction of p53 with DNA has been determined via a DNA-intercalating fluorescent probe. Furthermore, fluorescence resonance energy transfer (FRET) process have been utilized to probe the interaction between p53 and DNA in which p53 acts as a donor and DNA labeled with DMACA acts as an acceptor. The above method is simple, sensitive and provides insight into the biological functions of p53.更多还原