以牛血清白蛋白为靶五种污染物毒性评价新方法的研究
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摘要
环境污染问题依然是目前最重要的全球性问题之一。环境中的各种污染物对生态环境造成破坏的同时,也严重威胁着人类的生存与健康。污染物可以通过呼吸道吸入、食物链经消化道摄入和皮肤接触等渠道进入生命体内,通过与组成生命体的各种生物大分子(蛋白质、核酸等)发生作用造成生命体组织器官的损伤。因此研究环境中污染物对功能性生物大分子的毒性作用机理是非常必要的。关于污染物毒性研究,目前的研究方法多以生物毒性实验为主,但该方法很难从分子水平上解释污染物对机体的毒性作用机理。
蛋白质是生命体的物质基础,是生命体各种功能的直接执行者,其变性将导致生命体的功能或结构损伤,导致很多疾病的产生。环境中的很多的小分子污染物进入机体后都能够对机体内的蛋白质产生毒性作用从而使其变性和功能的丧失。因此,研究小分子环境污染物对蛋白质的毒性作用一直是环境污染与健康领域的热门课题。
本论文选择牛血清白蛋白(BSA)为靶分子,利用共振光散射光谱、荧光光谱、紫外.可见吸收光谱、圆二色性光谱、透射电镜等方法或手段研究了五种代表性污染物的毒性作用机理,建立了其对机体毒性作用研究的新方法。文中选择的五种污染物除纳米银外都是离子型污染物,实验制得的纳米银胶体本身也带有电荷,这样有助于污染物毒性的规律性探讨。
本论文选择的五种污染物除纳米银外都是离子型污染物,实验制得的纳米银胶体本身也带有电荷,这样有助于污染物毒性的规律性探讨。
本论文共分六章内容,具体如下:
第一章:介绍了主要的污染物毒性评价方法、靶分子蛋白质的概况、污染物毒性的相关研究进展和主要实验方法。
第二章:主要利用共振光散射技术研究了阴离子表面活性剂十二烷基苯磺酸钠(SDBS)在pH条件调控下对BSA的毒性作用,发现pH是控制SDBS使BSA中毒变性与解毒复性的重要条件,并结合其它方法对其机理进行了深入的探讨。
第三章:通过共振光散射技术在分子水平上研究了偶氮染料酸性嫩黄G与阳离子表面活性剂十六烷基三甲基溴化铵(CTMAB)组成的复合污染物对BSA的毒性作用机理,并考查了相关影响因素。研究发现两种污染物在单独与蛋白质作用时的共振光信号很弱,其复合物污染物与蛋白质作用时的共振光信号明显增强,这表明BSA-酸性嫩黄G-CTMAB的复合体系形成了三元离子缔合物。随后用吸收光谱、圆二色性光谱谱、透射电镜等方法对其毒性作用机理进行了深入的研究。
第四章:通过荧光光谱、同步荧光光谱、紫外吸收光谱、圆二色性光谱等光谱学方法研究了pb~(2+)对牛血清白蛋白(BSA)的毒性作用机理。研究结果表明pb~(2+)首先与BSA的骨架基团发生作用,使BSA骨架结构发生变化,内部疏水区域的色氨酸(Trp)等芳香环氨基酸逐步暴露出来,这在荧光光谱中表现为BSA内源荧光随Pb~(2+)的加入而发生猝灭。直到pb~(2+)浓度超过1.0x10~(-4) mol/L BSA的变性程度达到最大,之后pb~(2+)还与暴露在外面的Trp发生作用,进一步使荧光猝灭。
第五章:运用光谱学方法从分子水平上研究了纳米银(nanoAg)对BSA的毒性作用模式,结果表明nanoAg对BSA存在明显的损伤作用:nanoAg诱使BSA分子二级结构中α-螺旋比例下降,骨架结构变得松散;同时使得BSA内部疏水区的Trp等芳香环氨基酸的暴露增强,BSA自身的特征荧光明显猝灭。当nanoAg与BSA的配比达到1:96(以质量计)时,nanoAg对体系荧光光谱和共振光散射光谱的影响趋于稳定。共振光散射光谱、TEM、圆二色性光谱和电泳实验的研究结果表明范德华力和静电力的作用使BSA破坏了nanoAg表面的双电层平衡结构并包覆于nanoAg表面,生成新的带负电的BSA-nanoAg复合物。
第六章:最后对本论文的研究部分进行了总结,并分析了这种新的毒性评价今后的发展方向。
本论文丰富了污染物毒性作用机理的研究,为污染物对机体危害的防治提供了参考。
Environmental pollution is still one of the most important global problems at present. The environmental contaminants not only create destructions to the world ecology, but also threaten human survival and health seriously. The contaminants can enter organisms through respiratory tract inhalation, digestive tract intake and skin contact, and then damage the organs and tissues by interaction with biological macromolecule (protein, nucleic acid and so on). Thereforem, study on the mechanism of the contaminants toxicity to functional biological macromolecules is very essential. In this field, biological toxicity experiment is primary research technique, but this method is difficult to explain the toxicity mechanism of contaminants to biological macromolecules.
Protein is an important functional molecule in vivo, and its denaturation may cause functional or structural damage to life body. The main reason of many diseases (such as cataract, mad cow disease and diabetes) is protein denaturation. Recent research shows that contaminants in environment have toxic effect on organism protein and lead to protein denaturation. Therefore, study on toxicity mechanism of environment contaminants to protein has been a hot topic in field of environmental pollution and healthy.
In the research, we studied the toxicity mechanism of five typical contaminants targeted to bovine serum albumin (BSA) in molecule level by the methods of resonance light scattering (RLS) spectroscopy, fluorescence spectroscopy, ultraviolet-visible (UV-vis) absorption spectroscopy, circular dichroism (CD) spectroscopy and transmission electron microscopy, and a new method for evaluating the toxicity of contaminants is founded.
The five contaminants choosen in the research are all ionic compounds, except nanoAg. But nanoAg prepared in our study aslo has electric charges. So this paper is helpful to the regularity research of contaminants toxicity.
This study has six chapters, just as follows:
Chapter1: The main contaminants toxicity evaluation methods, the survey for target protein molecular, the related research about contaminants toxicity and the experiment techniques are introduced in this part.
Chapter2: By using the technique of resonance light scattering (RLS), we studied the toxicity mechanism of anionic surfactant sodium dodecyl benzene sulfonate (SDBS) targeted to bovine serum albumin (BSA) and found pH value was an important factor controlling the interaction. Furthermore, we made an in-depth research on the mechanism of the interaction by the techniques of UV absorption spectra, transmission electron microscopy (TEM) and circular dichroism spectra (CD).
Chapter3: By using the technique of resonance light scattering (RLS), we studied the toxicity of the complex system composed by cetyl trimethyl ammonium bromide (CTMAB) and acid flavine G targeted to bovine serum albumin (BSA).The results show that the blended system increasing transformation of RLS spectrum proves this conclusion which joining cationic surfactant CTMAB and acid flavine G in the BSA solution could form ionic aggregation, while Protein structure has also undergone a marked change. Furthermore, we made an in-depth research on the mechanism of the interaction by the techniques of UV absorption spectra, transmission electron microscopy (TEM) and circular dichroism spectra (CD).
Chapter4: By using the spectroscopic techniques of fluorescence spectroscopy, synchronous fluorescence spectroscopy, UV absorption spectroscopy, circular dichroism (CD) spectroscopy, we studied the toxicity mechanism of Pb~(2+) targeted to bovine serum albumin (BSA) in vitro condition. Pb~(2+) can cause the fluorescence quenching of BSA, which indicated that lead changed the skeleton group of BSA and caused the stepwise exposure of aromatic amino acid residues (Trp, Tyr, Phe) in the internal hydrophobic region of BSA. When the concentration of Pb~(2+) was up to 1.0×10~(-4)mol/L, the BSA would be completely denatured. The excess lead ion would react to the aromatic amino acid residues of BSA exposed in the impregnant, which leaded to the enhanced fluorescence quenching.
Chapter5: By spectroscopy techniques, the toxicity mechanism of nanoAg to organism was thoroughly studied from the level of bovine serum albumin (BSA) molecule. Results showed that nanoAg had obvious toxic effects to BSA: nanoAg can increase the helix and decrease the Beta, leading to the skeleton structure of BSA loose;; the exposal of internal hydrophobic amino acids enhanced, the characteristics fluorescence of BSA was obviously quenches. When the ratio of nanoAg and BSA increased to 1:96 (calculate in quality), the impacts of nanoAg to the fluorescence spectroscopy and resonance light scattering spectroscopy went to stabile. The result of resonance light scattering spectrum, TEM, circular dichroism spectra and electrophoresis experiments showed that BSA had destroyed the double-layer structure and covered in the surface, then generated to the BSA-nanoAg complex body because of the force among moleculars and electrostatic force.
Chapter6: Conclude the research parts and analyze the development direction of this new method for contaminants toxicity evaluation.
This study has enriched the research of contaminants toxicity mechanism, and provided some reference gists for contaminants toxicity prevention and treatment.
蛋白质是生命体的物质基础,是生命体各种功能的直接执行者,其变性将导致生命体的功能或结构损伤,导致很多疾病的产生。环境中的很多的小分子污染物进入机体后都能够对机体内的蛋白质产生毒性作用从而使其变性和功能的丧失。因此,研究小分子环境污染物对蛋白质的毒性作用一直是环境污染与健康领域的热门课题。
本论文选择牛血清白蛋白(BSA)为靶分子,利用共振光散射光谱、荧光光谱、紫外.可见吸收光谱、圆二色性光谱、透射电镜等方法或手段研究了五种代表性污染物的毒性作用机理,建立了其对机体毒性作用研究的新方法。文中选择的五种污染物除纳米银外都是离子型污染物,实验制得的纳米银胶体本身也带有电荷,这样有助于污染物毒性的规律性探讨。
本论文选择的五种污染物除纳米银外都是离子型污染物,实验制得的纳米银胶体本身也带有电荷,这样有助于污染物毒性的规律性探讨。
本论文共分六章内容,具体如下:
第一章:介绍了主要的污染物毒性评价方法、靶分子蛋白质的概况、污染物毒性的相关研究进展和主要实验方法。
第二章:主要利用共振光散射技术研究了阴离子表面活性剂十二烷基苯磺酸钠(SDBS)在pH条件调控下对BSA的毒性作用,发现pH是控制SDBS使BSA中毒变性与解毒复性的重要条件,并结合其它方法对其机理进行了深入的探讨。
第三章:通过共振光散射技术在分子水平上研究了偶氮染料酸性嫩黄G与阳离子表面活性剂十六烷基三甲基溴化铵(CTMAB)组成的复合污染物对BSA的毒性作用机理,并考查了相关影响因素。研究发现两种污染物在单独与蛋白质作用时的共振光信号很弱,其复合物污染物与蛋白质作用时的共振光信号明显增强,这表明BSA-酸性嫩黄G-CTMAB的复合体系形成了三元离子缔合物。随后用吸收光谱、圆二色性光谱谱、透射电镜等方法对其毒性作用机理进行了深入的研究。
第四章:通过荧光光谱、同步荧光光谱、紫外吸收光谱、圆二色性光谱等光谱学方法研究了pb~(2+)对牛血清白蛋白(BSA)的毒性作用机理。研究结果表明pb~(2+)首先与BSA的骨架基团发生作用,使BSA骨架结构发生变化,内部疏水区域的色氨酸(Trp)等芳香环氨基酸逐步暴露出来,这在荧光光谱中表现为BSA内源荧光随Pb~(2+)的加入而发生猝灭。直到pb~(2+)浓度超过1.0x10~(-4) mol/L BSA的变性程度达到最大,之后pb~(2+)还与暴露在外面的Trp发生作用,进一步使荧光猝灭。
第五章:运用光谱学方法从分子水平上研究了纳米银(nanoAg)对BSA的毒性作用模式,结果表明nanoAg对BSA存在明显的损伤作用:nanoAg诱使BSA分子二级结构中α-螺旋比例下降,骨架结构变得松散;同时使得BSA内部疏水区的Trp等芳香环氨基酸的暴露增强,BSA自身的特征荧光明显猝灭。当nanoAg与BSA的配比达到1:96(以质量计)时,nanoAg对体系荧光光谱和共振光散射光谱的影响趋于稳定。共振光散射光谱、TEM、圆二色性光谱和电泳实验的研究结果表明范德华力和静电力的作用使BSA破坏了nanoAg表面的双电层平衡结构并包覆于nanoAg表面,生成新的带负电的BSA-nanoAg复合物。
第六章:最后对本论文的研究部分进行了总结,并分析了这种新的毒性评价今后的发展方向。
本论文丰富了污染物毒性作用机理的研究,为污染物对机体危害的防治提供了参考。
Environmental pollution is still one of the most important global problems at present. The environmental contaminants not only create destructions to the world ecology, but also threaten human survival and health seriously. The contaminants can enter organisms through respiratory tract inhalation, digestive tract intake and skin contact, and then damage the organs and tissues by interaction with biological macromolecule (protein, nucleic acid and so on). Thereforem, study on the mechanism of the contaminants toxicity to functional biological macromolecules is very essential. In this field, biological toxicity experiment is primary research technique, but this method is difficult to explain the toxicity mechanism of contaminants to biological macromolecules.
Protein is an important functional molecule in vivo, and its denaturation may cause functional or structural damage to life body. The main reason of many diseases (such as cataract, mad cow disease and diabetes) is protein denaturation. Recent research shows that contaminants in environment have toxic effect on organism protein and lead to protein denaturation. Therefore, study on toxicity mechanism of environment contaminants to protein has been a hot topic in field of environmental pollution and healthy.
In the research, we studied the toxicity mechanism of five typical contaminants targeted to bovine serum albumin (BSA) in molecule level by the methods of resonance light scattering (RLS) spectroscopy, fluorescence spectroscopy, ultraviolet-visible (UV-vis) absorption spectroscopy, circular dichroism (CD) spectroscopy and transmission electron microscopy, and a new method for evaluating the toxicity of contaminants is founded.
The five contaminants choosen in the research are all ionic compounds, except nanoAg. But nanoAg prepared in our study aslo has electric charges. So this paper is helpful to the regularity research of contaminants toxicity.
This study has six chapters, just as follows:
Chapter1: The main contaminants toxicity evaluation methods, the survey for target protein molecular, the related research about contaminants toxicity and the experiment techniques are introduced in this part.
Chapter2: By using the technique of resonance light scattering (RLS), we studied the toxicity mechanism of anionic surfactant sodium dodecyl benzene sulfonate (SDBS) targeted to bovine serum albumin (BSA) and found pH value was an important factor controlling the interaction. Furthermore, we made an in-depth research on the mechanism of the interaction by the techniques of UV absorption spectra, transmission electron microscopy (TEM) and circular dichroism spectra (CD).
Chapter3: By using the technique of resonance light scattering (RLS), we studied the toxicity of the complex system composed by cetyl trimethyl ammonium bromide (CTMAB) and acid flavine G targeted to bovine serum albumin (BSA).The results show that the blended system increasing transformation of RLS spectrum proves this conclusion which joining cationic surfactant CTMAB and acid flavine G in the BSA solution could form ionic aggregation, while Protein structure has also undergone a marked change. Furthermore, we made an in-depth research on the mechanism of the interaction by the techniques of UV absorption spectra, transmission electron microscopy (TEM) and circular dichroism spectra (CD).
Chapter4: By using the spectroscopic techniques of fluorescence spectroscopy, synchronous fluorescence spectroscopy, UV absorption spectroscopy, circular dichroism (CD) spectroscopy, we studied the toxicity mechanism of Pb~(2+) targeted to bovine serum albumin (BSA) in vitro condition. Pb~(2+) can cause the fluorescence quenching of BSA, which indicated that lead changed the skeleton group of BSA and caused the stepwise exposure of aromatic amino acid residues (Trp, Tyr, Phe) in the internal hydrophobic region of BSA. When the concentration of Pb~(2+) was up to 1.0×10~(-4)mol/L, the BSA would be completely denatured. The excess lead ion would react to the aromatic amino acid residues of BSA exposed in the impregnant, which leaded to the enhanced fluorescence quenching.
Chapter5: By spectroscopy techniques, the toxicity mechanism of nanoAg to organism was thoroughly studied from the level of bovine serum albumin (BSA) molecule. Results showed that nanoAg had obvious toxic effects to BSA: nanoAg can increase the helix and decrease the Beta, leading to the skeleton structure of BSA loose;; the exposal of internal hydrophobic amino acids enhanced, the characteristics fluorescence of BSA was obviously quenches. When the ratio of nanoAg and BSA increased to 1:96 (calculate in quality), the impacts of nanoAg to the fluorescence spectroscopy and resonance light scattering spectroscopy went to stabile. The result of resonance light scattering spectrum, TEM, circular dichroism spectra and electrophoresis experiments showed that BSA had destroyed the double-layer structure and covered in the surface, then generated to the BSA-nanoAg complex body because of the force among moleculars and electrostatic force.
Chapter6: Conclude the research parts and analyze the development direction of this new method for contaminants toxicity evaluation.
This study has enriched the research of contaminants toxicity mechanism, and provided some reference gists for contaminants toxicity prevention and treatment.
引文
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