【作者】 余章；

【导师】 訾言勤；

【作者基本信息】 淮北师范大学 ， 分析化学， 2010， 硕士

【摘要】 摘要:蛋白质是由许多氨基酸组成的生物大分子,是生命的最基本物质之一,并在生命现象和生命过程中起着决定性的作用。其中人血清白蛋白(Human Serum Albumin,简称HSA)是人体内含量最丰富的运输蛋白,是由585个氨基酸残基组成的单肽链蛋白质,分子量约为66500,主要起维持血液的正常渗透压和运送亲水分子的作用,广泛应用于金属离子、药物药理和毒理研究、污染毒物的危害机理及基因变异研究等方面。而胰蛋白酶(Trypsin)是一种动物来源的蛋白水解酶,其单一肽链含有233个氨基酸残基和6对二硫键,分子量为24000,专一作用于精氨酸、赖氨酸的羧基所形成的肽键,广泛应用于医药、食品、工业领域以及蛋白质的序列分析、亲和、吸附、分离等方面。寻找一种简单、快速、高灵敏度的测量蛋白质的方法是科学家目前关注的焦点,同时随着测定蛋白质各种形式探针的日益增多,蛋白质与探针之间的相互作用的研究变得尤为重要。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以及发展高选择性、高灵敏度的蛋白质定量分析新方法,在临床医学、生命科学、毒理学及药代动力学上具有重要意义,该领域已成为从事临床医学、化学和生命科学等学科研究的科研工作者共同关注的课题之一。本论文利用吸收光谱技术和共振光散射技术研究了胰蛋白酶-染料体系、辅酶Ⅰ-染料体系和人血清白蛋白-染料体系,探讨了染料与胰蛋白酶、辅酶Ⅰ、人血清白蛋白的作用机理,建立了利用光学探针测定胰蛋白酶、辅酶Ⅰ及人血清白蛋白的新方法。论文共分五个部分。一乙基紫和胰蛋白酶的相互作用及其含量测定在中性介质中,阳离子碱性染料乙基紫与大分子胰蛋白酶的强烈相互作用,导致了分子间的构象发生变化,从而引起分子光谱最大吸收值的变化。应用光谱法研究了反应体系的酸度及用量、染料乙基紫的用量、反应时间等影响反应因素,确定了反应的最佳实验条件,建立了测定胰蛋白酶的可行、简便、快捷的方法。并推导了染料乙基紫对胰蛋白酶的作用机理。实验的结果表明:运用分光光度法测得的结果更为满意。最佳反应条件为pH=7.0,缓冲溶液用量为2.0 mL,乙基紫的用量为2.0 mL,反应时间约20 min,反应现象比较明显,在胰蛋白酶的含量为2.0～130.0 mg/L的浓度范围内成线性关系。运用测量结果对样品进行分析,并测定回收率。得到的结果较为满意。二磷酸盐和胰蛋白酶的相互作用及其含量测定在B-R缓冲介质中,邻甲苯胺亚磷酸盐与大分子胰蛋白酶的相互作用,导致了分子间的构象发生变化,从而引起分子光谱最大吸收值的变化。应用光谱法研究了反应体系的酸度及用量、邻甲苯胺亚磷酸盐的用量、反应时间等影响反应因素,确定了反应的最佳实验条件,建立了测定胰蛋白酶的可行、简便、快捷的方法。实验的结果表明:最佳反应条件为pH=10.88,缓冲溶液最佳用量为2.0 mL,邻甲苯胺亚磷酸盐的最佳浓度为100 mg/L,反应时间约20 min左右最为稳定,反应现象比较明显,在胰蛋白酶的含量为15.0 mg/L～240.0 mg/L的浓度范围内成线性关系。运用测量结果对样品进行分析,并测定回收率。用于猪胰脏中胰蛋白酶含量的测定,结果满意。三亚甲蓝和胰蛋白酶的相互作用及其含量测定本章研究了生物染料亚甲兰在阴离子表面活性剂十二烷基硫酸钠的存在下,与胰蛋白酶相互作用的共振光散射光谱。研究发现在pH为3.78的酸性条件下,亚甲兰与阴离子表面活性剂的共振光散射强度较弱,加入胰蛋白酶后其共振光散射信号大大增强,且与胰蛋白酶的浓度在2.0～14.0 mg/L范围呈良好的线性关系,据此建立了用有机染料为探针测定胰蛋白酶含量的共振光散射新方法。该方法灵敏度高,检出限为0.634 mg/L。四乙基紫和辅酶Ⅰ的相互作用及其含量测定本章用紫外-可见分光光度法研究了乙基紫(EV)与辅酶Ⅰ(NAD)的相互作用,并对影响体系的因素进行了探究。结果表明,在pH=4.78的条件下,NAD的加入使EV在其最大吸收峰(595 nm)处吸光度增强,比EV溶液自身的吸光度显著增大。用紫外-可见分光光度计测定,在595 nm处EV-NAD溶液的吸光度值与EV溶液的吸光度值之差与NAD的浓度在一定范围内有线性关系,线性响应范围为0.05-80.0 mg/L,检出限为0.0271 mg/L,用于辅酶Ⅰ的测定,结果满意,由此建立起快速测定NAD的实用方法。同时,初步探讨了EV-NAD的结合机理。五乙基紫和人血清白蛋白的相互作用及其含量测定本章研究了乙基紫(EV)与人血清白蛋白(HSA)的结合反应。在pH=3.78的条件下,HSA的加入使EV在其最大吸收峰(595 nm)处吸光度增强,比EV溶液的吸光度显著增大。用紫外-可见分光光度计测定,在595 nm处EV-HSA溶液的吸光度值与EV溶液的吸光度值之差ΔA与HSA的浓度c在一定范围内有线性关系,线性响应范围为5.0～150.0 mg/L,检出限为0.0971 mg/L,用于人血清白蛋白样品测定结果满意,由此建立起快速测定HSA的实用方法。该法具有简便、快速、干扰少、灵敏度高的特点。同时,初步探讨了EV-HSA结合机理。更多还原

【Abstract】 Abstract: The protein is the biological macromolecule of which is composed many amino acids, is one of life’s most basic materials, and is playing the decisive role in the biological phenomena and the life process. Human Serum Albumin (HSA) is the most abundant carrier protein in blood circulation. It is Single peptide protein consisting of 585 Amino acid residues, whose molecular weight approximately is 66500. It mainly plays a role of a carrier for diverse substances. What’s more, it is largely responsible for maintaining normal osmolarity in the plasma. It is used in medicine such as shok, edema and hypoproteinemia. It is important to study the interaction of small molecules and metallic ion with the protein because protein-drug binding plays an important role in pharmacology, pharmacodynamics, toxicology and gene mutation. And Trypsin is a kind of proteolytic enzymes from animal. The single peptide contains 233 amino acid residues and 6 pairs of disulfide bond, whose molecular weight approximately is 24000. It affects on peptide linkage which is formed by arginine and lysine of carboxyl. It widely applies in the medicine, food, the industrial field as well as the aspects of the protein sequential analysis, compatible, adsorption, separation and so on.The scientists focus on finding a simple, quick and high sensitive method to determine the protein. There are more and more probes for determination of protein. To study the interaction between protein and probes become very important. Researching the binding mechanism and process of toxic materials and drugs with proteins and investigating novel quantification method of proteins with high selectivity and sensitivity have many importances in clinical medicine, life sciences, toxicology and pharmacokinetics. Thus, it has been an interesting research field of clinical medicine, chemistry and life sciences.This thesis studies the Trypsin-dye system and HSA-dye system, discusses the interaction mechanism of dye with Trypsin and HSA and applies to the determination using resonance light scattering technique and absorption spectral technique. The thesis is divided into five chapters. 1. Investigation on Interaction of Trypsin with Ethyl Violet Dye and its applicationThe interaction of Ethyl violet (EV) with Trypsin in pH=7.0 Britton-Robinson buffer solution has been investigated by UV/Vis spectrophotometry technique. The increase in the absorption at 595 nm was proportional to the concentration of Trypsin base on EV, providing a basis for the quantitative determination of Trypsin. The absorbance of each solution at 595 nm was measured and the regression equation wasΔA=0.28412+0.00151c (mg/L). The linear range for the determination of Trypsin was from 2.0 to 130.0 mg/L with correlation coefficient of 0.9991 and the detection limit was 0.0869 mg/L(3σ). This method was simple and efficient than the ordinary methods and had been applied to the direct determination of Trypsin with satisfactory results.2. Ya-o-toluidine phosphate of the determination of trypsin researchB-R in the buffer solution, the sub-o-toluidine bisphosphonates trypsin and macromolecular interaction between the elements led to a change in conformation, which led to the molecular absorption spectra of the largest value. Application spectrometry system of reaction and the amount of acidity, the sub-o-toluidine amount of bisphosphonates, such as reaction time affect the reaction, a reaction to determine the best experimental conditions for the establishment of the determination of trypsin feasible, simple and quick Methods. The experimental results show that: the best conditions for the reaction pH = 10.88, amount to 2.0 mL, sub-o-toluidine bisphosphonates for the amount of 2.0 mL, the reaction time about 20 minutes or so of the most stable, the response is more obvious in the pancreas The protein content of 15.0 ~ 240.0 mg / L of concentration within the framework of a linear relationship. Use measurements of samples for analysis and determination of recovery. More satisfied with the results.3. A Study on Interaction of Trypsin with Methylene Blue and its applicationThe interaction of Methylene Blue (MB) with Trypsin in pH= 3.78 Britton-Robinson buffer solution (B-R) has been investigated in the presence of sodium dodecyl sulfate (SDS) by UV/Vis spectrophotometry and resonance Rayleigh-scattering (RRS) technique. In acidic medium, SDS and MB can combine to Trypsin to form a macromolecular complex, causing the great enhancement of RRS intensity at 467.2 nm. Moreover, the interaction mechanism was also discussed. It was showed that the anionic surfactant SDS can firstly react with Trypsin via electrostatic attractions, hydrophobic bonds and hydrophilic bonds to form SDS-Trypsin aggregation. Then MB molecules bind to the aggregation by hydrogen bonds to form the SDS-Trypsin-MB complex, which results in the great enhancement of RRS intensity. The intensity of RRS of each solution at 467.2 nm was measured and the regression equation wasΔI=-28.0196+55.41263c (mg/L). The linear range for the determination of Trypsin was from 2.0 to 14.0 mg/L with correlation coefficient of 0.993 5 and the detection limit was 0.634 mg/L(3σ). This method was simple and efficient than the ordinary methods and had been applied to the direct determination of Trypsin with satisfactory results.4. Investigation on Interaction of NAD with Ethyl Violet Dye and its applicationThe interaction of ethyl violet (EV) withβ-Nicotinamide adenine dinucleotide (NAD) in pH=4.78 Britton-Robinson buffer solution has been investigated by UV/Vis spectrophotometry technique.The increase in the absorption at 595 nm was proportional to the concentration of NAD base on EV,providing a basis for the quantitative determination of NAD.The absorbance of each solution at 595 nm was measured and the regression equation wasΔA=0.01714+0.02225c (mg/L).The linear range for the determination of NAD was from 0.05 to 80.0 mg/L with correlation coefficient of 0.999 0 and the detection limit was 0.0271 mg/L(3σ).This method was simple and efficient than the ordinary methods and had been applied to the direct determination of NAD with satisfactory results.5. Research About Ethyl Violet and Human Serum AlbuminThe interaction of ethyl violet (EV) with human serum albumin (HSA) in pH=3.78 Britton-Robinson buffer solution has been investigated by UV/Vis spectrophotometry technique.The increase in the absorption at 595 nm was proportional to the concentration of HSA base on EV,providing a basis for the quantitative determination of HSA.The absorbance of each solution at 595 nm was measured and the regression equation wasΔA=0.1736+0.0015c(mg/L).The linear range for the determination of HSA was from 5.0 to 150.0 mg/L with correlation coefficient of 0.999 7 and the detection limit was 0.0971 mg/L(3σ).This method was simple and efficient than the ordinary methods and had been applied to the direct determination of HSA with satisfactory results.更多还原