【作者】 王柱命；

【导师】 宋正华；

【作者基本信息】 西北大学 ， 分析化学， 2010， 博士

【摘要】 蛋白质是生命的物质基础,是生命科学的重要研究对象。蛋白质与小分子物质之间的相互作用研究有助于考察蛋白质的结构和功能与小分子配体的结构和性质之间存在的相关性,并已成为生命科学、化学和临床医学研究领域的重要研究课题。本论文采用流动注射-化学发光法,以溶菌酶、牛血清白蛋白、过氧化氢酶和肌红蛋白对化学发光试剂鲁米诺的增敏作用为基础,分别建立了鲁米诺-溶菌酶、鲁米诺-牛血清白蛋白、鲁米诺-过氧化氢酶和鲁米诺-肌红蛋白稳态化学发光体系；以溶菌酶和牛血清白蛋白为模型蛋白,探讨了蛋白质与头孢菌素之间的相互作用并做了富有创新性的研究工作,首次建立了蛋白质-药物相互作用的流动注射-化学发光法模型,并且成功用于四种模型蛋白与头孢菌素等药物相互作用及分析应用研究。论文内容分为两部分：第一部分综述总结了分子光谱法进展及其应用于研究蛋白质与小分子物质相互作用的现状,引用文献282篇(第一章)。第二部分研究报告1.蛋白质-药物相互作用的流动注射-化学发光法模型的构建。溶菌酶(或牛血清白蛋白)与鲁米诺在线生成1：1的二元复合物,能够增强鲁米诺化学发光,以此建立了稳态的鲁米诺-溶菌酶(或鲁米诺-牛血清白蛋白)化学发光体系；以头孢菌素与鲁米诺-溶菌酶(或鲁米诺-牛血清白蛋白)的相互作用生成1：1：1三元复合物并能猝灭鲁米诺-溶菌酶(或鲁米诺-牛血清白蛋白)化学发光为基础,构建了蛋白质-药物相互作用的流动注射-化学发光法模型并推导出计算作用参数的公式：lg(I0-I)／I=lgKD+nlg[D](第二、三章)。2.蛋白质-药物相互作用的流动注射-化学发光法模型的应用。运用本论文建立的蛋白质-药物相互作用模型,获得了溶菌酶-头孢菌素和牛血清白蛋白-头孢菌素的作用参数,同时用荧光猝灭法进行了验证,两种方法得到的结果基本一致。大部分头孢菌素类药物与蛋白相互作用的结合常数KD均在103-105水平,表明该类药物与溶菌酶或牛血清白蛋白之间存在较强相互作用。结合能力大小遵循以下顺序：头孢哌酮、头孢曲松、头孢噻肟>头孢呋辛、头孢克洛>头孢羟氨苄、头孢拉定、头孢唑啉,表明随着药物代次的升高,药物与蛋白质之间结合越来越强。结合位点数n约为1.0,表明头孢菌素在溶菌酶或牛血清白蛋白分子中的结合位点都是一个,并可能位于溶菌酶分子中Trp62或牛血清白蛋白分子中Trp212氨基酸残基附近。热力学参数均为：ΔG<0,ΔH>0,ΔS>0,表明溶菌酶或牛血清白蛋白同头孢菌素药物相互作用形成复合物的过程是自发进行的,并主要以疏水作用力相结合(第二、三章)。头孢菌素药物与过氧化氢酶能够发生相互作用,对鲁米诺-过氧化氢酶体系的化学发光有猝灭作用,据此运用流动注射-发学发光法研究了过氧化氢酶与头孢菌素的相互作用。运用蛋白质-药物作用模型,计算了过氧化氢酶与头孢菌素相互作用的的结合常数及结合位点数。除第一代头孢菌素外,二、三代药物与过氧化氢酶的结合常数KD均在104-105水平,表明它们之间存在强的亲合作用；结合位点数n约为1.0,表明药物在过氧化氢酶分子中有一个结合位点。本方法还成功用于口服头孢拉定胶囊中的药物含量测定和人体尿液中头孢拉定的监测,口服500.0 mg头孢拉定后,2小时后头孢拉定的排出量达到最大值,8小时内总代谢率为79.51%,计算得到头孢拉定总的代谢常数(K)和半衰期(t1／2)分别为0.4822和1.45(第四章)。基于鲁米诺-肌红蛋白体系强烈的化学发光能被头孢菌素药物抑制,运用流动注射-化学发光法研究了肌红蛋白与头孢菌素药物的相互作用,获得了作用参数。本方法也应用于头孢克洛胶囊中的含量测定及口服头孢克洛后人体尿液中代谢状况监测。药物含量测定的回收率范围为93.0%-106.5%,测定值与标示量接近；尿液监测的结果为1.5小时头孢克洛的排出量达到最大值,8小时内总代谢率为51.75%,总的代谢常数(K)和半衰期(t1／2)分别为0.8063和0.86(第五章)。应用鲁米诺-肌红蛋白化学发光体系,研究了肌红蛋白与三聚氰胺的相互作用,建立了测定纳克级三聚氰胺的化学发光分析方法。鲁米诺-肌红蛋白体系化学发光强度的减小值与三聚氰胺浓度的对数值在0.01 ng mL-1-50ng mL-1的范围内呈现良好的线性关系,检出限为3 pg mL-1(3σ),测定的相对标准偏差小于3.0%(n=7)。该方法已经成功应用于测定乳制品中的三聚氰胺,回收率为93.4-106.5%(第六章)。以氯雷他定对鲁米诺-肌红蛋白体系化学发光反应的抑制作用为基础,研究了肌红蛋白和氯雷他定的相互作用,建立了测定氯雷他定的化学发光分析方法。氯雷他定浓度的对数值与化学发光强度降低值成线性关系,线性浓度范围为0.1 ngmL-1-100.0 ng mL-1,检出限为0.03 ng mL-1(3σ),实验测定的相对标准偏差均小于3.0%(n=7)。该方法获得了肌红蛋白-氯雷他定结合的作用参数,并成功应用于药片、尿液及血清样品中氯雷他定的测定(第七章)。更多还原

【Abstract】 Protein is the essential biological material and basilic studying object of life science. Study on the interaction between protein and small molecules has been the current important subject in the fields of biology, chemistry and clinical medicine as it can help to explore the relativity of the structure and nature of small molecules with structure and function of protein. In this thesis, based on the CL of luminol can be significantly enhanced by lysozyme, BSA, catalase and myoglobin, the steady CL system of luminol-lysozyme, luminol-BSA, luminol-catalase and luminol-myoglobin was proposed. Using lysozyme and BSA as the model proteins, the interaction of protein with cephalosporin was investigated by FI-CL method and a FI-CL model for studying the interaction of protein-drug was constructed for the first time. The proposed model was applied successfully to the interaction study of four model proteins with drugs. The main content consisted of two parts:Part I:ReviewThe current development of molecular spectroscopy was summarized and the research status for the interaction between protein and small molecules by molecular spectroscopy were described.282 references was cited (in Chapter 1).Part II:Research reports1. Constructing of FI-CL model for studying the interaction of protein-drug. Lysozyme (or BSA) and luminol could react to form 1:1 complex on line, which could greatly enhance the luminol CL intensity, then the steady CL system of luminol-lysozyme (or luminol-BSA) was proposed. Based on cephalosporin binding to luminol-lysozyme (or luminol-BSA) formed 1:1:1 complex and remarkable quenched the CL of luminol-lysozyme (or luminol-BSA), the FI-CL model of protein-drug interaction was constructed and the formula lg(Io-I)/I=lgKD+nlg[D]was deduced (in Chapter 2 and 3).2. Applications of FI-CL model for studying the interaction of protein-drug. Using the proposed model, the interaction parameters of lysozyme-cephalosporin and BSA-cephalosporin were calculated. Meanwhile, the binding parameters were also studied by fluorescence quenching method. The results obtained by the proposed model agreed well with the results obtained by fluorescence quenching method illuminated that the proposed model would be a feasible method in the study of protein-drug interaction. The binding constant KD values of the drugs mostly were at 103-105 level suggesting that there was high binding affinity of cephalosporin to lysozyme or BSA. The binding ability of the studied cephalosporin drugs followed the pattern:cefoperazone, ceftriaxone and cefotaxime> cefuroxime and cefaclor> cefadroxil, cefradine and cefazolin, which consisted with that of their antibacterial ability. The number of binding potential point n approximately equaled to 1.0 indicating that there was one class of binding site to cephalosporin analogues in lysozyme or BSA and cephalosporin probably bound to the site of Trp62 in lysozyme or the active site near Trp212 in BSA. The calculated thermodynamic parameters were△G＜0,△H>0 and△S> 0, which meant that the binding process was spontaneous and the hydrophobic effect was the major binding force in the interaction of lysozyme or BSA with cephalosporins (in Chapter 2 and 3). Based on the inhibitory effect of cephalosporin on the luminol-catalase CL system, a new method for studying the interaction of catalase with cephalosporin was constructed. Utilizing the proposed protein-drug interaction model, the binding constant and the number of binding potential point were calculated. The results indicated that there was high binding affinity and one class of binding site of cephalosporin in catalase. The calculated thermodynamic parameters showed that catalyse bound with cephalosporins by hydrophobic effect. The proposed procedure was applied successfully to determine cefradine in capsules and monitor the excretion of cefradine in human urine samples. It was found that the excretive cefradine concentration reached its maximum after orally administrated for 2 hours, the cefradine excretive ratio in 8 hours was 79.51%, the total elimination rate constant (K) and the half-life time (t1/2) of cefradine in the body were 0.4822 and 1.45, respectively (in Chapter 4). Based on the inhibitory effect of cephalosporin on the luminol-myoglobin system, the interaction of myoglobin with cephalosporin was studied by FI-CL and the interaction parameters were obtained. The proposed method was applied successfully to the determination of cefaclor in capsules with the recoveries range from 93.0%-106.5%, and the results agreed well with the labeled amount. The proposed method was also applied to monitoring the excretion of cefaclor in human urine samples. It was found that the excretive cefaclor concentration reached its maximum after orally administrated for 1.5 hours, the cefaclor excretive ratio in 8 hours was 51.75%, the total elimination rate constant (Κ) and the half-life time(t1/2) of cefaclor in the body were 0.8063 and 0.86 hours, respectively (in Chapter 5). The interaction of myoglobin with melamine was investigated using luminol-myoglobin CL system. A sensitive chemiluminescence method for the determination of melamine was presented based on the inhibitory effect of melamine on the CL reaction between luminol and myoglobin, and the decrement of CL intensity was proportional to the concentration of melamine ranging from 0.01 to 50.0 ng mL"1 with the detection limit of 3 pg mL-1 (3σ). The proposed method was applied successfully to the determination of melamine in milk products, and the recovery was from 93.4 to 106.5%(in Chapter 6). Based on the inhibitory effect of loratadine on the luminol-myoglobin chemiluminescence reaction, the interaction between myoglobin and loratadine was studied and a CL method for the determination of loratadine was presented. The decrement of CL signal was linear with the logarithm of loratadine concentration over the range from 0.1 to 100.0 ng mL-1 with the detection limit of 0.03 ng mL-1 (3a) and relative standard deviation of less than 3.0%(n= 7). The proposed procedure was applied successfully to obtaining the interaction parameters of myoglobin-loratadine complex and determining loratadine in the medicine of tablets, human serum and urine (in Chapter 7).更多还原