水中多环芳烃与DNA之间的相互作用及机制

发布时间：2019-05-17 10:51

【摘要】：多环芳烃(PAHs)被美国环保署(EPA)列为优先控制的一类持久性有机污染物。由于具有致癌、致畸和致突变性,且普遍存在于污染土壤、水体和空气中,PAHs的环境行为受到国内外广泛关注。众多资料表明,PAHs能被生物有机体吸收并在组织间分配;PAHs与生物质之间的相互作用已成为环境领域研究的热点之一。目前,物理作用支配下,在分子尺度上PAHs与生物质间相互作用如何?该研究开始受到关注;其中,可否利用荧光分析技术来揭示DNA与PAHs之间的相互作用?国内外相关资料仍很缺乏。本论文以菲和芘为PAHs代表物,利用荧光分析技术探究了 PAHs与DNA之间的相互作用及机制;利用荧光光谱分析技术和荧光猝灭滴定技术,研究了不同酸度情况下(酸度影响DNA的质子化过程)PAHs与DNA之间的结合规律,分析了质子化反应对DNA-PAHs结合能力的影响。主要研究结果如下:(1)利用荧光分析技术,研究了 DNA与菲和芘之间的相互作用。两种PAHs分别被DNA溶液滴定,通过激发-发射荧光矩阵光谱,测定了光谱强度变化和波长位移。结果证明,菲和芘能与DNA中碱基单元结合。然而,由于菲和芘之间的分子尺寸效应,两者反应的程度和机制存在差异。DNA能够显著导致菲荧光发射光的猝灭,然而其对芘的荧光猝灭效应较弱。菲与DNA通过一个作用位点、浓度单位上以1/1的方式进行结合,但由于DNA-芘分子间相互作用力非常弱,芘与DNA的结合能力较弱。进一步研究发现,虽然菲芘与DNA之间结合能力存在差异,但所增加的DNA均能够造成菲和芘发射波谱的蓝移。这表明DNA和菲、芘之间均形成不发光的复合物。该研究结果为揭示DNA和PAHs分子之间的物理性相互作用提供了一种新手段。(2)DNA能够通过静态猝灭的方式引起PAHs分子的的荧光猝灭,主要原因为二者之间形成了不产生荧光的复合物;在此基础之上,进一步探究了不同酸度影响下PAHs与DNA之间的结合规律。通过分析发射光谱的变化发现,随着酸度降低,DNA能明显地导致菲发射光谱蓝移,证实酸度能造成DNA碱基的质子化,从而可能影响PAHs与碱基之间的π-π电子堆积。进一步研究发现,在酸度较强的条件下,DNA不能有效地造成菲荧光猝灭,表明DNA分子外围的磷酸基质子化阻碍了菲与DNA内部碱基的结合。计算了荧光猝灭常数,得出猝灭常数遵循pH 9.0(0.34)≈ pH 7.0(0.36)pH3.0(0.26)的规律。结合位点的研究结果揭示,三种酸度条件下,DNA和菲的结合位点均是一,表明酸度条件并不影响DNA与菲的结合位点,仅通过碱性条件下脱质子化增强DNA与菲的结合效率(结合常数)。本论文研究结果为寻求PAHs分子猝灭剂、评估PAHs的生物分子毒理、揭示PAHs与生物遗传物质之间的分子作用等提供了重要基础依据。
[Abstract]:Polycyclic aromatic hydrocarbons (PAHs) are classified as a class of persistent organic pollutants by the United States Environmental Protection Agency (EPA). The environmental behavior of PAHs is of great concern at home and abroad due to the carcinogenic, teratogenic and mutagenicity, and is ubiquitous in the polluted soil, water and air. Many data indicate that PAHs can be absorbed by biological organisms and distributed among tissues; the interaction between PAHs and biomass has become one of the hot spots in the field of environment research. At present, how is the interaction between PAHs and biomass at the molecular scale under the control of physical action? The study has begun to be of concern; in which, can fluorescence analysis techniques be used to reveal the interaction between DNA and PAHs? The relevant information at home and abroad is still lacking. In this paper, the interaction and mechanism between PAHs and DNA were studied by means of fluorescence analysis. The fluorescence spectrum analysis and the fluorescence quenching titration were used to study the interaction and mechanism between PAHs and DNA. The binding of PAHs and DNA in the condition of different acidity (acidity-affected DNA) was studied, and the effect of protonation on the binding capacity of DNA-PAHs was analyzed. The main results are as follows: (1) The interaction between DNA and phenanthrene and phenanthrene is studied by fluorescence analysis. The two kinds of PAHs were titrated by DNA solution respectively, and the spectral intensity and the wavelength shift were measured by the excitation-emission fluorescence matrix. As a result, the phenanthrene and the antigen can be combined with the base unit in the DNA. However, due to the molecular size effect of the phenanthrene, there is a difference in the degree and mechanism of the reaction. DNA can significantly lead to the quenching of the fluorescence emission of the phenanthrene, but its fluorescence quenching effect on the fluorescence is weak. The interaction between the DNA and the DNA is very weak, and the binding ability of the DNA to the DNA is weak due to the very weak interaction between the DNA and the DNA. Further studies have found that, although the binding capacity between the phenanthrene and the DNA is different, the increased DNA can cause the blue shift of the phenanthrene emission spectrum. This indicates that no light-emitting complex is formed between the DNA and the phenanthrene. The results of this study provide a new means to reveal the physical interaction between the DNA and the PAHs molecules. (2) The fluorescence quenching of the PAHs in the DNA can be caused by the static quenching, and the main reason is that the fluorescence-free complex is formed between the two, and the binding rule between the PAHs and the DNA under the influence of different acidity is further explored. By analyzing the change of the emission spectrum, with the decrease of the acidity, the DNA can obviously lead to the blue shift of the phenanthrene emission spectrum, and it is confirmed that the acidity can cause the protonation of the DNA base, which may affect the electron-to-electron accumulation between the PAHs and the base. The further study found that, under the condition of strong acidity, the DNA could not effectively cause the fluorescence quenching of the phenanthrene, indicating that the protonation of the phosphate group on the periphery of the DNA molecule hindered the binding of the phenanthrene to the internal base of the DNA. The fluorescence quenching constant was calculated, and the law of pH 9.0 (0.34) and pH 7.0 (0.36) pH 3.0 (0.26) was obtained. The results of the study revealed that the binding sites of DNA and phenanthrene in three kinds of acidity condition are one, indicating that the acidity condition does not affect the binding sites of the DNA and the phenanthrene, and the binding efficiency (binding constant) of the DNA and the phenanthrene is enhanced only by deprotonation under alkaline conditions. The results of this paper are to find the molecular-quenching agent of PAHs, to evaluate the biological molecular toxicology of PAHs, and to reveal the molecular function of PAHs and the biological genetic material.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X52;X17

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