【作者】 董立军；

【导师】 陈兴国；

【作者基本信息】 兰州大学 ， 分析化学， 2007， 博士

【摘要】 蛋白质是生命科学的重要研究对象之一，其中人血清白蛋白（HSA）是人体内含量最丰富的运输蛋白。HSA有许多重要的生理学和药理学功能，能与许多内源和外源性物质如脂肪酸、氨基酸、荷尔蒙、阴阳离子和药物等结合，起到存储和转运作用。人免疫球蛋白（γ-G）作为淋巴细胞的细胞表面受体在许多细胞活动中扮演着重要角色，同时在人体免疫应答中发挥着至关重要的作用。因此，HSA和γ-G已成为研究最广泛的目标蛋白。发展高灵敏度、高选择性的蛋白质定量分析新方法以及研究毒性物质、药物等小分子配体与蛋白质的相互作用机理和作用过程，在生命科学、临床医学、毒理学及药代动力学上具有重要意义，该领域已成为从事生命科学、化学和临床医学等学科研究的科研工作者共同关注的课题之一。本论文基于研究蛋白质的定量分析新方法及小分子与蛋白质相互作用的重要意义及国内外研究趋势，在总结前人工作的基础上，综合利用共振光散射光谱、荧光光谱、紫外-可见吸收光谱、傅里叶变换红外光谱、圆二色性光谱、计算机分子模拟技术和动态激光散射技术，从以下几方面进行了创新性研究：1、将自行组装的全内反射及流动注射装置与荧光光谱仪联用，建立了测定人血清样品中蛋白质含量的新方法，获得了与兰州大学第一医院一致的结果。2、研究了偶氮胂-TB、钙试剂两种染料和中药活性组分大豆甙元与蛋白质的相互作用及结合机理，根据Scatchard方程和Stern-Volmer方程计算了反应的结合常数和结合位点数，进而根据van’t Hoff方程求得反应的热力学参数，讨论了上述有机小分子与蛋白质的主要作用力类型。3、利用傅立叶变换红外光谱技术和圆二色性光谱技术考察了蛋白质与有机小分子作用前后其二级结构的变化，并且采用计算机分子对接模拟技术研究了染料与人血清白蛋白的键合，提出了染料与人血清白蛋白相互作用的分子模型，获得与实验一致的结果。4、研究了人免疫球蛋白在油包水型微乳液中与药物大豆甙元的相互作用，并且与在水溶液中的相互作用进行了比较，同时利用荧光偏振技术和动态激光散射技术研究了微乳液水池大小对人免疫球蛋白和大豆甙元相互作用的影响。论文共分为七章：第一章：简要介绍了蛋白质的结构、功能和性质；综述了蛋白质的定量分析方法及有机小分子与蛋白质相互作用的研究内容和研究方法。第二章：基于测定水／四氯化碳（H₂O／CCl₄）界面的全内反射共振光散射（TIR-RLS）强度，建立了人血清样品中蛋白质的定量分析新方法。在pH 4.10且有十六烷基溴化钠（CTAB）存在的条件下，偶氮胂-TB与人血清白蛋白（HSA）的相互作用可导致体系的TIR-RLS信号增强，最大散射峰位于330nm处，增强的TIR-RLS强度与HSA浓度在0.5-42.3μg／mL范围内成正比，检测限（3σ）为198ng╱mL。该方法已用于6个人血清样品中蛋白质含量的测定，其最大相对标准偏差为1.2％，回收率在97-103％之间。第三章：基于在pH 4.1的酸性条件下，蛋白质对甲基蓝（MB）的共振光散射（RLS）信号有强烈增强作用的现象，建立了测定蛋白质的流动注射-共振光散射新方法。在333nm处增强的RLS强度与蛋白质浓度成正比。该体系中人血清白蛋白（HSA）和牛血清白蛋白（BSA）的线性范围分别为2.0-37.3μg／mL和1.0-36.0μg／mL，检测限（3σ）分别为45ng／mL和80ng╱mL。该方法已成功地用于5个人血清样品中蛋白质含量的测定，其最大相对误差不超过为1％，回收率在98-102％之间，进样率为60h^-1。第四章：基于在pH 4.1的酸性溶液中，蛋白质对酸性铬兰K（ACBK）的共振光散射（RLS）信号有强烈增强作用的现象，建立了测定人血清样品中蛋白质含量的流动注射-共振光散射新方法。在264 nm处增强的RLS强度分别与人血清白蛋白（HSA）和牛血清白蛋白（BSA）的浓度在2.0-40.0μg／mL和4.0-33.8μg／mL范围内成正比，检测限（3σ）分别为85ng╱mL和124 ng／mL。该方法己成功地用于6个人血清样品中蛋白质含量的测定，其最大相对标准偏差不超过为2％，回收率在97-103％之间，进样率为60 h^-1。第五章：利用共振光散射技术、傅立叶变换红外光谱法和圆二色性光谱法研究了染料偶氮胂-TB（ATB）与人血清白蛋白（HSA）之间的相互作用。在最佳实验条件下，计算了不同温度下ATB与HSA相互作用的结合常数及结合位点数，根据热力学参数确定了ATB与HSA之间的作用力类型，并定量分析了ATB与HSA的相互作用对HSA二级结构的影响。研究结果表明，结合常数及最大结合位点数随着温度的升高而降低。由热力学数据可知，ATB与HSA之间的结合是自发的、放热的过程，并且以疏水作用为主；ATB的结合能使HSA的α-螺旋结构减少，β结构增加。第六章：利用共振光散射技术、傅立叶变换红外光谱法、圆二色性光谱法和分子模拟技术研究了染料钙试剂与人血清白蛋白（HSA）之间的相互作用。光谱数据表明，钙试剂与HSA相互作用的结合常数及最大结合位点数随着温度的升高而降低。由热力学数据可知，钙试剂与HSA之间的结合是自发的、放热的过程，并且以疏水作用为主。对HSA二级结构的研究表明，钙试剂的结合能使HSA的α-螺旋及β-转角结构减少、β-折叠结构增加。分子模拟结果显示钙试剂结合在HSA的IIA子域内。第七章：利用荧光光谱法、紫外-可见吸收光谱法、圆二色性光谱法和动态激光散射技术研究了模拟生理条件下在水／AOT／异辛烷的反相微乳介质中大豆甙元与人免疫球蛋白（γ-G）之间的相互作用。光谱数据表明，大豆甙元通过静态机理猝来γ-G的内在荧光，以单一结合部位与γ-G相互作用，其结合常数随着温度的升高而降低，大豆甙元与γ-G在微乳介质中的相互作用要比水溶液中的相互作用强有力。由热力学数据可知，大豆甙元与γ-G之间的结合是自发的、放热的过程，并且以疏水作用为主。CD光谱表明，γ-G与大豆甙元的结合使其二级结构发生了变化。更多还原

【Abstract】 Protein is one of the most important substances investigated in life sciences. Human Serum Albumin （HSA） is the most abundant carrier protein in blood circulation. It has many important physiological and pharmacological functions, which can bind many exogenous and endogenous ligands in blood, and realize transport and distribution of many molecules and metabolites, such as fatty acids, amino acids, hormones, ions and many diverse drugs. Human Immunoglobulin （γ-G）, as cell surface receptor of lymphocyte, plays important roles in many cell actions, it also plays a key role in human immune response. Therefore, HSA andγ-G have been the most extensively studied proteins. Investigating novel quantification method of proteins with high sensitivity and selectivity and researching the binding mechanism and process of toxic materials and drugs with proteins have many importances in life sciences, clinical medicine, toxicology and pharmacokinetics. Thus, it has been an interesting research field of life sciences, chemistry and clinical medicine. In this dissertation, on the basis of the previous research, the resonance light scattering technique, fluorescence spectroscopy combined with UV-visible absorption, Fourier transform infrared （FT-IR）, circular dichroism （CD） spectroscopy along with computational modeling were used to investigate the novel quantification method of proteins and research the interaction of several organic small molecule substances with proteins. The following major innovative works were carried out:1. The total internal reflection and flow injection combined with resonance light scattering technique were used to quantify the protein in human serum samples. The homemade instruments of total internal reflection and flow injection were connected with spectrofluorophotometer. Protein in human serum samples were determined using the combination techniques mentioned above, the results were consistent with that ones obtained by the first Hospital of Lanzhou University.2. The interaction and binding mechanism of Arsenazo-TB, Cal-Red and Daidzein withproteins were investigated, the binding parameters including binding constants and the number of binding sites were calculated according to Scatchard and Stern-Volmer equation, the thermodynamic parameters were also calculated according to van’t Hoff equation and the main interaction force between organic small molecules and proteins was discussed.3. The alternations of proteins’ secondary structure caused by the binding of smallligands were estimated by Fourier transform infrared （FT-IR）, circular dichroism （CD） spectroscopic methods. The computational modeling method was used to study the dye-HSA interaction, the molecular model of the interaction of dye and HSA was obtained and the results were consistent with the experimental results.4. The interaction of Daidzein withγ-G in water/oil microemulsions has been investigated and compared with that of in aqueous solution, the influence of the size of water pool in microemulsions on the interaction of Daidzein withγ-G was also investigated by fluorescence polarization and dynamic laser scattering technique.This dissertation consists of seven chapters:Chapter 1: The structures, functions and natures of proteins were briefly described. The methods of determination of proteins and the study of interaction of organic small ligands with proteins were reviewed.Chapter 2: A novel quantification method of protein in human blood serum samples was proposed based on the measurement of total internal reflected resonance light scattering （TIR-RLS） at water/tetrachloromethane （H₂O/CCl₄） interfaces. At pH 4.10 and in the presence of Cetyltrimethylammonium bromide （CTAB）, the interaction of Arsenazo-TB （ATB） with human serum albumin （HSA） results in strong enhanced TIR-RLS signals with the maximum spectra peak located at 330 nm. The enhanced TIR-RLS intensity was found to be proportional to the concentration of HSA in the range of 0.5-42.3μg/mL and its limit of determination （3σ） is 198 ng/mL. The content of HSA in six blood serum samples was determined with the maximum RSD of 1.2% and the recovery of 97-103%.Chapter 3: A novel method of flow injection sampling combined with resonance light scattering detection was developed for the determination of total protein. This method is based on the enhancement of RLS signals from methyl blue （MB） by protein at pH 4.1. The enhanced RLS intensities at 333 nm were proportional to the protein concentration over the range of 2.0-37.3μg/mL and 1.0-36.0μg/mL for human serum albumin （HSA） and bovine serum albumin （BSA）, respectively. The corresponding limits of detection （3a） of 45 ng/mL for HSA and 80 ng/mL for BSA were attained. The method was successfully applied to the quantification of total proteins in five human serum samples, the maximum relative error was less than 1% and the recovery was between 98%-102%, The sample throughput was 60 h^-1.Chapter 4: A novel method of flow injection sampling combined with resonance light scattering detection was developed for the determination of protein concentration in human serum samples. This method is based on the enhanced RLS signals of proteins binding with acid chrome blue K （ACBK） at acidic aqueous solution of pH 4.10. The enhanced RLS intensities at 264 nm were proportional to the proteins concentration over the range of 2.0-40.0μg/mL and 4.0-33.8μg/mL for human serum albumin （HSA） and bovine serum albumin （BSA）, respectively. The corresponding limits of detection （3σ） of 85 ng/mL for HSA and 124 ng/mL for BSA were attained. This method was successfully applied to the quantification of total proteins in six human serum samples, the maximum relative standard deviation was less than 2% and the recovery was between 97%-103% for standard addition method, the sample throughput was 60 h^-1.Chapter 5: The interaction between Arsenazo-TB and human serum albumin （HSA） was studied by Resonance light scattering （RLS）, Fourier transformed IR （FT-IR） and circular dichroism （CD） spectroscopic methods. The binding parameters of Arsenazo-TB with HSA were studied at different temperatures under the optimum conditions. It is indicated that the binding parameters decreased with the increasing of the temperature. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy was hydrophobic interaction. The alterations of protein secondary structure in the presence of Arsenazo-TB were quantitatively calculated with reductions ofα-helix and with increases ofβstructures.Chapter 6: The interaction between Cal-Red and human serum albumin （HSA） was studied using Resonance light scattering （RLS）, Fourier transformed Infrared （FT-IR）, Circular dichroism （CD） and molecular modeling methods. The binding parameters of HSA with Cal-Red were studied at different temperatures and it is indicated that the binding constant K and the maximum binding number N decreased with increasing the temperature. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy was hydrophobic interaction. The alterations of protein secondary structure in the presence of Cal-Red were quantitatively calculated with reductions ofα-helix andβ-turn structures and with increases ofβ-sheet structure. The primary binding site of Cal-Red was located in IIA subdomain of HSA.Chapter 7: The interaction of Daidzein withγ-G in reverse microemulsions of H₂O/AOT/isooctane at simulative physiological pH has been investigated by fluorescence, UV-absorption, Circular dichroism （CD） spectroscopic methods and dynamic laser scattering technique. The results of spectroscopic measurements suggested that the intrinsic fluorescence ofγ-G was quenched by Daidzein through static quenching mechanism and the interaction was via a single class of binding site. The binding constant K decreased with increasing the temperature and the binding force in emulsion was larger than that of in aqueous solution. The binding process was exothermic and spontaneous, as indicated by the thermodynamic analyses, and the major part of the binding energy was hydrophobic interaction. The result of CD spectroscopy suggested that the secondary structure ofγ-G changed after interacted with Daidzein.更多还原