蛋白质标记荧光探针的光谱研究及分析应用

【作者】 孙洋；

【导师】 樊君；

【作者基本信息】 西北大学 ， 化学工程， 2012， 博士

【摘要】 蛋白质是组成生物体的基本物质,维持其新陈代谢活动的正常进行。蛋白质构造和功效的研究及定量分析将为我们研究生命在不同条件下的变化机制奠定基础,同时也对临床医学、生命科学及药物化学等学科的发展具有重要的意义。蛋白质标记荧光探针日益广泛地应用于生物分析、蛋白质组学中的识别、结构变化的研究及各项生理活动指标检测等。因此开发新型蛋白质标记荧光探针、建立选择性好、灵敏度高的蛋白质定量检测分析方法已成为临床医学、生物化学及生命科学等学科研究人员共同努力的方向。本论文共分为四章：第一章,简要介绍了蛋白质的结构功能,综述了研究蛋白质标记荧光探针的种类、标记原理、定量分析蛋白质的方法及应用研究进展。第二章,采用荧光及吸收光谱研究了荧光素钠(SF)与人血清白蛋白(HSA)的相互作用,根据Stern-Volmer、Scatchard及Van’t Hoff等相关方程分别计算了猝灭常数、结合常数、结合位点数及热力学参数,确定了HSA对SF的猝灭机理及作用方式,根据能量转移理论求得SF与HSA的结合距离及能量转移率,采用同步、三维荧光光谱研究了SF在结合过程中对HSA构象的影响；实验中发现,加入一定范围浓度的HSA会显著猝灭SF的荧光,且光强猝灭程度与HSA浓度成正比,据此建立测定血清蛋白的方法,线性范围为0.15～15×10-7M,方法具有高灵敏度,检测极限为1.46×10-8M；最后,考察体系pH值的变化和不同共存物质对于蛋白质定量分析结果的影响,利用SF作为荧光探针,在测定人血清中血清蛋白总量的实验中,其结果与G-250法基本一致。第三章,采用溶剂化法、量子力学的二阶摄动理论计算新型1,8-萘酰亚胺类荧光探针分子量子产率、偶极矩等光物理参数；利用密度泛函理论Density Functional Theory(DFT)及Amsterdam Density Functional (ADF)计算软件讨论探针分子发光机理；应用光谱分析技术对探针分子与HSA的相互作用进行了详细的研究：根据Stern-Volme、Scatchard及Van’t Hoff方程计算并讨论了结合参数、反应热力学参数及结合作用力类型；通过取代位点竞争实验,确定了探针分子在HSA中的结合位点；利用同步荧光光谱及圆二色谱考察了探针分子对HSA次级结构及构象的影响；HSA的加入会猝灭探针分子荧光,在一定范围内猝灭程度与HSA浓度成正比,据此,首次建立以1,8-萘酰亚胺类荧光探针测定蛋白的新方法,以新型1,8-萘酰亚胺衍生物作为荧光探针测定人血清中血清蛋白总量的结果与G-250法基本一致。第四章,首次利用分子模拟技术和光谱法详细研究了麦角甾4,6,8,22-四烯-3酮(麦角甾酮,国家一类新药,ergone)及土大黄苷(RA)与HSA的相互作用,通过分子对接软件确定ergone和RA与HSA相互作用的结合区域和结合模式。光谱研究结果表明,ergone和RA与HSA的结合作用猝灭了HSA的内源性荧光并生成了疏水性的复合物；利用荧光猝灭相关公式和差热量热仪计算了结合反应的结合常数、结合位点数及热力学参数；三维、同步荧光光谱及CD数据揭示了ergone和RA对HSA次级结构含量的影响。采用溶剂化法、量子力学的二阶摄动理论计算ergone和RA的光物理参数,利用DFT理论及ADF软了探讨了两种药物的发光机理；基于血清蛋白对ergone和RA荧光光谱具有良好的响应行为,在模拟生理条件下以ergone和RA为荧光探针,建立了一种灵敏的蛋白质定量分析方法,HSA线性响应浓度范围为10-8～10-6M,检测限(3δ)均达到10-9M,考察了金属离子对检测结果的影响。以ergone和RA作为荧光探针测定人血清中血清蛋白总量的结果与G-250法基本一致。本论文基于蛋白质标记荧光探针的研究及分析应用趋势,综合利用光谱分析技术及分子模拟计算软件,建立了新的定量分析方法,研究的创新性如下：1.以新型1,8-萘酰亚胺类衍生物、RA和ergone为荧光探针,采用量子力学的二阶摄动理论计算探针分子的光物理参数,利用密度泛函理论(DFT)及ADF软件研究了探针分子发光机理；2.综合利用光谱分析技术(荧光光谱、吸收光谱、圆二色谱(CD))研究了SF、新型1,8-萘酰亚胺类衍生物、RA和ergone探针分子与HSA的相互作用,根据Stern-Volmer、 Scatchard和Van’t Hoff方程探讨了探针分子与HSA的结合机理、结合力类型以及结合位点等问题；3.通过计算机分子模拟技术考察了药物分子与HSA的作用模式,构建了结合模型,为利用药物分子光谱特性进行蛋白质标记提供了理论参考；采用三维、同步荧光光谱及圆二色谱考察了药物分子在结合过程中对HSA构象的影响,检测、定量分析了药物分子结合后蛋白质二级结构的变化；4.通过荧光探针标记蛋白质后光物理参数变化实现对蛋白质生理活动的检测,建立蛋白质定量分析新方法,该方法具有线性响应范围广(10-8～10-6M)和灵敏度高(检测限为10-10M)的特点。本法用于人血清样品中血清蛋白总量的分析结果与G-250法和医院提供数据基本一致。更多还原

【Abstract】 Protein which maintains the metabolism of the organism is fundamental elements of life. The research on protein structure and function, and the quantitative analysis would illustrate the change in life under the physiological or pathological consideration, which is important for the clinical diagnosis and drug screening. The applications of protein marker fluorescent probes in biological analysis and proteomics become more extensive recently. The probes are used to recognize and detect the complicated conformational changes of proteins. Developing protein marker fluorescent probes and novel quantification method of proteins with high sensitivity and selectivity is important for the field of life sciences, chemistry and clinical medicine. This dissertation consists of four chapters.Chapter1:The structures, functions of proteins were briefly introduced firstly. The developments of protein marker fluorescent probes were reviewed, and the kinds of probes, marked mechanism and methods of proteins quantitative analysis were summarized.Chapter2:The interactions between sodium fluorescein (SF) and human serum albumin (HSA) were investigated by fluorescence spectroscopy. According to the modified Stern-Volmer equation, the association constants (Ka) between SF and HSA at three different temperatures were obtained to be1.34×107,1.00×107,0.80×107M-1, respectively. It is proved that the fluorescence quenching of SF by HSA is a result of the formation of SF-HSA complex. The thermodynamic parameters, enthalpy change (△H) and entropy change (AS) for the reaction were calculated to be-23.29kJ mol-1and57.09J mol-1K-1according to van’t Hoff equation, indicating that the hydrophobic force was the dominant intermolecular force in stabilizing the complex. The distance r between donor (SF) and acceptor (HSA) was obtained to be5.1nm according to Forster’s non-radioactive energy transfer theory. The effect of SF on the conformation of HSA was also analyzed using3D fluorescence spectroscopy and synchronous fluorescence spectroscopy. The fluorescence intensity of SF-HSA complex was proportional to the concentration of HSA, based on which, a new quantitative assay of protein was presented. The linear range was0.015～1.5μM, and the sensitivity of the method was high with detection limit was determined to be1.5×10-9M. The effects of pH and interfering substance on the detection were also investigated. The results indicated that the most of the water-soluble amino acids, metal ion and antioxidant do not interfer or only interfer slightly under the permission of±5.0%relative error, whereas SDS, Lecithin, APG, Cu2+, Fe3+, Sucrose and Tryptophane produced obvious interference. Determining serum in human serum by this method gived results which were very close to those obtained by Coomassie Brilliant Blue colorimetry.Chapter3:The photophysical parameters of probes such as quantum yield and dipole moments were calculated by salvation and quantum mechanics of second order perturbation theory. The luminiferous mechanism of probes was investigated by Density Functional Theory (DFT) and Amsterdam Density Functional (ADF) soft. According to the equations of Stern-Volmer、Scatchard and Van’t Hoff, the binding parameters, binding force and binding thermodynamics parameters were calculated. Using the displacement experiments, the binding site of probes was illustrated. The effect of probes on the second structure of protein has been studied by the synchronous fluorescence and CD spectroscopy. The fluorescence of probes could be quenched by the addition of serum albumin, and the intensity of probes was linear with the concentrations of protein, based on which, a new fluorometric method for detecting protein using1,8-naphthalimide derivatives as fluorescent probes was developed. Determination of protein in human serum by this method gave results which were very close to those obtained by using Coomassie Brilliant Blue colorimetry.Chapter4:The interactions of rhapontin (RA) and ergosta-4,6,8(14),22-tetraen-3-one (ergone) with HSA were investigated by the molecular modeling and spectrua comprehensively for the first time, which applied the information about the binding domain and binding model of RA and ergone in HSA. The spectral results showed that the fluorescence of HSA was quenched by the binding of drugs, and the hydrophobic complexes were formed. The binding constant, number of binding site and thermodynamic parameters of binding interaction were calculated by different scanning calorimeter (DSC) and fluorescence spectroscopy. Three-dimensional and synchronous fluorescence spectra revealed the effect of three drugs on the second structure of HSA. Moreover, the photophysical parameters of RA and ergone were calculated by salvation and quantum mechanics of second order perturbation theory, and the luminiferous mechanism was investigated by the DFT and ADF soft. Ergone exhibited a remarkable fluorescence enhancement (EE) for serum albumin, while RA exhibited a fluorescence quenching for serum albumin, based on which, the novel sensitive quantitative determination of proteins were developed by using ergone and RHA as fluorescence probes under the physical condition. Good calibration curves of the proteins were obtained in the range of10-8～10-6M, with detection limits (3σ) of10-9M, and the effect of metal ions on the detection results were investigated. Detecting total proteins in human serum by this method gave results which were close to those obtained by using Coomassie Brilliant Blue colorimetry.In this dissertation, on the basis of the previous research, using the spectral analysis technics along with computational modeling were used to investigate the protein marker fluorescent probes and novel quantification method of proteins. The following major innovative works were carried out:1. Using SF, the novel1,8-naphthalimide derivatives, RA and ergone which are gradients of natural drug as the fluorescent probes. The photophysical parameters were calculated with quantum mechanics of second order perturbation theory, and the mechanism of photo luminescence of probes was investigated with the Density Functional Theory and ADF soft.2. The interactions of probes with HSA were studied by the comprehensive spectral analysis technology, such as the fluorescence, absorption and CD spectrum. According to the equations of Stern-Volmer, Scatchard and Van’t Hoff, the binding mechanism, style of binding force and binding site was investigated.3. The interaction of RA and ergone with HSA was investigated by the molecular modeling, and the binding mode was developed, which provided the theoretical reference for the probes marked protein. The secondary structure compositions of HSA were estimated by quantitative analysis using synchronous fluorescence and CD spectroscopy selves deconvolution with second-derivative resolution enhancement and curve-fitting procedure.4. After labeling protein, the photophysical parameters of fluorescent probes changed remarkably, base on which, a new fluorometric method for detecting HSA in the HCl-Tris buffer solution (pH=7.4) was developed. The linear ranges of the calibration curves were10"8-10-6M for HSA, with the detection limits (3σ) of10-10M. The method has been applied to the determination of total serum albumin in human serum samples collected from the hospital and the results were in good agreement with those reported by the hospital and the method of G-250.更多还原