摘要
随着人类社会的发展,人们的生活水平得到了很大的提高,但同时环境污染对人类的生存和发展造成了严重威胁。环境问题虽越来越受到重视,但依然是目前最重要的全球性问题之一。人们通过空气、水、食物等接触到的潜在危险物越来越多。这些潜在的危险物质能通过消化道、呼吸道以及皮肤接触等渠道进入生物体内,通过各种方式与体内的大分子生命物质(如蛋白质、核酸、酶等)直接或间接地发生作用,影响或者破坏了这些分子的生物功能,对各个器官造成不同程度的损伤,从而对机体产生毒性效应,诱发各种疾病。因此,研究这些具有潜在威胁的环境污染物对生物大分子的毒性作用机理是非常必要的,这有助于对这些物质的毒性做出全面的评价,为制订合理的标准来保障人身体健康和安全提供参考和技术支持。
现在最常用的污染物毒性评价方式主要有动物实验和流行病学调查两种,但却都无法从分子水平上解释污染物的毒性作用机理,不能从根本上阐明毒性效应。为了弥补这一缺点,本论文从分子水平出发,在体外条件下采用光谱学手段探讨了小分子污染物与蛋白质功能大分子的毒性相互作用,有助于增强人们对该类化学污染物在血液传输过程与血清白蛋白结合后对白蛋白毒性作用的认识,为此类污染物的毒性作用的阐明提供了新的视角。
本论文选用了生物体内最普遍的蛋白质—血清白蛋白(SA)为靶分子,在体外条件下分子水平上,通过荧光光谱、同步荧光光谱、三维荧光光谱、紫外-可见吸收光谱、圆二色性光谱等技术手段研究了其与两种常见的水溶性偶氮染料的毒性作用机理,建立了一种评价污染物生命体毒性的新方法。另外,在实验的进行过程中发现并解决了内滤效应对荧光测定的影响,以及蛋白质特征光谱的解读等相关问题。
本论文共分六章内容,其中主要实验内容包括四部分:
第一部分:选用了酸性嫩黄G(AY11)作为研究对象,以牛血清白蛋白(BSA)为靶,利用荧光光谱、紫外可见光谱、圆二色谱等光谱学手段,从分子水平上研究了AY11对蛋白质分子的影响。实验发现,AY11能够与BSA发生中等强度的结合,会与BSA生理过程中与正常结合的内源与外源化合物相竞争,改变了蛋白质的结构,从而影响蛋白分子正常的运输作用,产生毒性作用。另外,通过实验证明了AY本身的吸收对荧光的测定结果影响很小,可以忽略。
第二部分:选用了柠檬黄(AY23)作为研究对象,选用了两种血清白蛋白(SA)牛血清白蛋白(BSA)和人血清白蛋白(HSA)为靶,利用上一章的荧光光谱、紫外可见光谱、圆二色谱等光谱学手段,并引入了更加直观的三维荧光光谱法,从分子水平上研究了AY23对蛋白分子的毒性作用影响。实验结果表明,柠檬黄对两种蛋白均具有毒性作用,使蛋白的结构均发生了不同程度的改变。
通过这两部分的研究,发现在光谱法研究蛋白质体系中存在的一系列问题,如荧光测定过程中的内滤效应对测定的影响,以及蛋白质光谱特征解释不够明确或存在偏差。因而在以下两部分部中进行了相关的研究。
第三部分:由于前两部分都采用了有颜色的偶氮染料作为污染物,而染料本身的颜色,会导致其对特定波长的光有吸收作用,这将有可能会干扰蛋白质荧光光谱的测定。为了确定这种吸收作用会不会对实验产生干扰,以及如何消除这种干扰,本部分进行了一系列的相关实验,确定了在前两部分的实验中这种干扰对实验的影响很小。但是在其他污染物与蛋白的体系研究中发现,这种干扰还是会对实验结果产生比较严重的影响,为此设计了一种装置和配套的方法来消除这种干扰对实验结果的影响,相关成果已经申请专利。
第四部分:在第二部分的试验中引入了三维荧光光谱技术,但是在图谱的解析过程中发现,现有的理论并不完善,甚至有些还存在偏差。因此,在本部分中,通过三维荧光光谱及紫外可见吸收光谱技术,利用用氨基酸来模拟蛋白质的方法研究了蛋白质的特定光谱,对蛋白质各个特征峰的产生机理做了详细的解释,为以后的分子水平研究提供了详细的参考。
The rapid development of society has not only improved the living standard of humans, but also poses great threat to the human health. In recent years, considerable attention has been paid to the globally environmental pollution. However, humans are still exposed to these persistent pollutants via air, water, foods and other ways, which would transport directly through the digestive tract, respiratory tract and skin contact channels into the biological body and produce the toxicity to humans by the direct or indirect interactions with biological macromolecules in vivo, such as proteins, nucleic acids, and enzymes. Therefore, it is of great importance to research on the potential toxicity of pollutants to the bimolecular, which will help more to complement studies on the environmental risk assessment of pollution, and provide some reference and technical support for the relevant standard of protecting human health and safety.
There are lots of toxicity evaluations for pollutants, of which the most commonly two are animal experiments and epidemiological investigations, but these methods can not explain the toxicity mechanism from the molecular level. In order to compensate for this, we adopted the spectroscopic techniques to study the toxicity mechanism in vitro from the protein level, which will help to understand and predict the toxicity of the contaminants to the protein in the blood, and it also provide a new perspective to clarify the toxicity of contaminants.
In this paper, the mechanism interactions of two common soluble azo dyes to serum albumins have been investigated under simulated physiolocigal condition by means of fluorescence, synchronous fluorescence, three-dimensional fluorescence, UV-vis absorption (UV), and circular dichroism (CD). We also designed a device to verify the inner filter effect by experiment and clarified the related issues about the spectral characteristics of proteins from a new perspective.
The paper is divided into six parts, of which the main experiments consist of four parts:
Part 1:We studied the effect of acid yellow (AY 11) exposure to the common protein bovine serum albumin (BSA) by several spectroscopic techniques including fluorescence, UV and CD. It was found that AY could interact with BSA moderately by competing with the endogenous and exogenous compounds to the protein binding, and it caused conformational changes of protein, which could affect its activity or even change its physiological function. Besides, AY was proved to have little impact on the fluorescence of BSA by a verification experiment.
Part 2:We selected two serum albumins (SA), human serum albumin (HSA) and BSA, as the targets to evaluate the toxic effects of tartrazine (AY23) by means of the above techniques and a more intuitive three-dimensional fluorescence spectroscopy from the protein level. And the results confirmed that AY23 indeed impact the conformation of both HSA and BSA.
A series of problems in the protein study were founded from these above parts, such as the inner filter effect to the fluorescence, and the unclearly explanation or deviation on the protein spectral characteristics. Therefore, these problems were studied in the next two sections.
Part 3:With their own colors, the dyes will cause the light absorption among the specific wavelengths, which may possible interfere with the protein fluorescence spectra. To determine the impact of dyes absorption, a series of experiments were made and the results proved that there was little effect on the fluorescence spectra because of the absorption. However, in other systems, the absorption has serious effects on the results of the protein fluorescence. Thus we designed a device to eliminate the impact of absorption, and the relevant results have been patented.
Part 4:The adoption of three-dimensional fluorescence spectroscopy in the second part revealed that some protein spectral characteristics were not explained perfectly and some deviations were founded in the explanation. As a result, in this section, we simulated the protein with the twenty amino acids to obtain the specific protein spectra by means of the three-dimensional fluorescence spectroscopy and UV methods. The improvement of the protein spectra explanation will provide a detailed reference to the future molecular study.
引文
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