【作者】 张珑；

【导师】 钟儒刚；

【作者基本信息】 北京工业大学 ， 生物医学工程， 2007， 硕士

【摘要】 在生物系统中有过亚硝酸根生成的发现,使过亚硝酸根(ONOO-)逐渐成为生物学、医学等领域关注的对象。过亚硝酸根是强氧化剂,能够引发自由基链反应和硝化芳环,并进一步损伤生物大分子,特别是硝化损伤酪氨酸和蛋白质酪氨酸残基,生成3-硝基酪氨酸。3-硝基酪氨酸的形成被认为是体内硝化产物形成的生物标志,3-硝基酪氨酸与炎症、心血管疾病和神经退行性疾病等多种病症相关。因此,研究过亚硝酸根对酪氨酸的硝化损伤有着重要意义。生物体内存在的金属离子可参与ONOO-介导的硝化反应过程,这不仅会加剧酪氨酸的损伤,更重要的是可能会提高相关疾病的发病机率。找到具有促进硝化损伤作用的金属离子,不仅可以进一步揭示过亚硝酸根的生物活性和反应机制,也可以了解金属离子的生物作用和3-硝基酪氨酸相关疾病的发病机制。以Co(II)、Cu(II)对过亚硝酸根硝化损伤酪氨酸和蛋白质酪氨酸残基的促进作用以及维生素C对Co(II)、Cu(II)促进作用的抑制作用的研究为本论文的主要内容,该研究结果对揭示ONOO-的生理作用机制,了解相关疾病的发病因素并进行预防或治疗有着重要意义。本论文采用经过本实验室改良的亚硝酸异戊酯与过氧化氢反应的方法,合成过亚硝酸盐溶液。检测硝化产物的紫外吸收,建立过亚硝酸根损伤酪氨酸和蛋白质酪氨酸残基的实验条件,选择合适的反应物初始浓度。利用紫外分光光度法以硝化产物生成量作为指标,研究七种常见金属离子(MgCl2,MnCl2,CaCl2,ZnSO4,CuSO4,CoCl2,NiCl2)对过亚硝酸根硝化损伤酪氨酸和色氨酸的影响;并进一步研究Co(II)、Cu(II)的浓度与其促进硝化损伤作用之间的关系;在此基础上研究维生素C对Co(II)、Cu(II)促进硝化的抑制作用。对Co(II)、Cu(II)促进ONOO-硝化纤维蛋白原和牛血清白蛋白上酪氨酸残基的作用进行研究;讨论Co(II)、Cu(II)的浓度与其促进硝化损伤作用之间的关系和维生素C对Co(II)、Cu(II)的促进硝化损伤的抑制作用,利用三维荧光法对上述结果进行验证。更多还原

【Abstract】 Since it was found that peroxynitrite(ONOO-) can be produced in biological systems, peroxynitrite has come to the wide attention in the field of biology and physics. Peroxynitrite, as a strong oxidant, can trigger free radical chain reaction and nitrify benzene. By the two ways, it damages multiplicate biological molecules, especially tyrosine and tyrosine residuals of proteins and nitriation products are produced. Now, nitrifying tyrosine is regarded as a most important biomarker of nitriation products in biological systems. And it has relations with a variety of pathophysiological conditions such as inflammation, cardiovascular and nerve diseases. So it is meaningful to research tyrosine nitration by peroxynitrite. Many kinds of metal ions exist in human, and some of them have positive effects on peroxynitrite nitrifying tyrosine. It will enhance risk of correlative ills. We tried to find out these metal ions. By doing this, not only biological activity and reaction mechanism of peroxynitrite were clearer but also functions of metal ions in organism and mechanisms of correlative ills with 3-Ntyr could be better known. One of the main contents of the paper was positive effects of Co(II)、Cu(II) to peroxynitrite nitrifying tyrosine and tyrosine residuals, the other concent was negative effects of Vitamin C to peroxynitrite’s nitriation with Co(II) and Cu(II). The study is meaningful to explain mechanism of ONOO- physiological action. It is also meaningful to understand arising factors of correlative ills and prevention or treat them.In this paper, we would use isoamyl nitrite and H2O2 to synthesize ONOO-. Through assaying ultraviolet absorption of nitration products, the experiments condition was to be established, and the suitable concentrations of reactants were selected. With ultraviolet spectrophotometric method and outputs of nitration products as targets, seven salts of metal ions (MgCl2,MnCl2,CaCl2,ZnSO4,CuSO4, CoCl2,NiCl2) were selected to assist in peroxynitrite damaging tyrosine and tryptophan. And the relation between Cu(II) and Co(II) positive effects and their concentration were to be studied. On base of that, we were to study Vitamin C negative effects to nitration with Cu(II) and Co(II). We were to study metal ions influence on nitration tyrosine residuals of fibrinogen and BSA by peroxynitrite. Cu(II) and Co(II) also had positive effects dependent on their concentrations. Vitamin C can resist positive effects of Cu(II) and Co(II). With 3-Dimentions fluorescence, damages of proteins were assayed, and the results approved above experiments data.更多还原