等温滴定量热法课件

1、(最新整理)等温滴定量热法2021/7/261等温滴定微量热法(Isothermal Titration Calorimetry, ITC)林杰ITC Isothermal titration calorimetry (ITC) is used to measure the heat adsorbed or released during changes in the composition of a systemundergoing a titration process. 等温滴定量热技术（ITC）是一种监测由结合成分的添加而起始的任 何化学反应的热力学技术，即用一种反应物滴定另一种反应物

2、，随着加入滴定剂的数量的变化，测量反应体系温度的变化2021/7/263historybuilt in the secondhalf of the 1960s to study chemical reactions.During the1970s, the sensitivity of instruments was in the range of mJ, and other first applications were developed such as the study of (metal + ligand) complexes 8 and the adsorption of arom

3、atic compounds by molecular sieves .In 1980s, the sensitivity was improved to J range. New applications in the 1980s were related to biology,biochemistry and physical chemistry such as the study of ligand binding processes and micelle formation灵敏度的提高2021/7/264historySince the beginning of 1990s, the

4、 number of published papers related to isothermal titration calorimetry has symptomatically increased due to rapid diffusion of new commercial calorimeters in the scientific community.2021/7/265historyDuring the decade of 1990, isothermal titration calorimetry has evolved from a specialist method to

5、 a widely used technique . During the 2000s, isothermal titration calorimetry was widely employed in the design and discovery of drugs and in addition to this, in this last decade an isothermal titration calorimeter with open cell was employed for the study of liquid mixtures .2021/7/266ITC2021/7/26

6、7在恒温下，注射器中的“配体”溶液滴定到包含“高分子”溶液的池中。当配体注射到池中，两种物质相互作用，释放或吸收的热量与结合量成正比。当池中的高分子被配体饱和时，热量信号减弱，直到只观察到稀释的背景热量。2021/7/268上图：横坐标：时间纵坐标：热功率峰底与峰尖之间的峰面积为每次注射时释放或吸收的总热量。下图：横坐标：滴定物与样品溶液的摩尔比纵坐标：滴定产生的总热量反应过程的结合等温曲线图形2021/7/269ITC可以直接测量焓变H，结合常数Ka，而不对反应体系产生影响，也不引入修饰基团，因此测得的结果更加可信典型的ITC数据配体溶液20次注射到ITC池的蛋白溶液中。每个注射峰（上图）下

7、方的区域与注射所释放的总热量相等。当这种综合的热量相对添加到池中的配体摩尔比作图时，就获得了相互作用的完整结合等温线（下图）。用单位点模型来验证数据。化学计量、结合常数及焓的数值都显示在框内。2021/7/2610流程1、确定合适的反应物浓度2、准备样品3、滴定收集数据4、校正后的数据非线性回归得到热力学参数5、分析模型1、微克级3、配体浓度大于大分子浓度1、考虑反应物性质2、尽量避免稀释热3、抽气处理1、设置对照组2、平行试验3、Origin软件2021/7/2611模型分析正确理解反应过程首先要理解模型给出的各种参数的正确意义。根据模型给出的各种热力学参数确定反应的相关性质。结合位点数不同

8、，平衡常数的物料平衡的表达公式是不同的2021/7/26122021/7/2613characteristics它通过高灵敏度、高自动化的微量量热仪连续、准确地监测和记录一个变化过程的量热曲线，原位、在线和无损伤地同时提供热力学和动力学信息，它已经成为鉴定生物分子间相互作用的首选方法。可获得生物分子相互作用的完整热力学参数，包括结合常数（Ka）、结合位点数（n）、摩尔结合焓（H）、摩尔结合熵（S）、摩尔恒压热容（Cp），和动力学参数(如酶促反应的Km和kcat）,用来表征生物分子间的相互作用。2021/7/2614独特特点：样品用量小，方法灵敏度和精确度高(仪器最小可检测热功率2 nW，最小可

9、检测热效应0.125uJ，生物样品最小用量0.4ug，温度范围2 - 80 ，滴定池体积(1.43 ml)。实验时间较短(典型的ITC实验只需30-60分钟，并加上几分钟的响应时间)，操作简单(整个实验由计算机控制，使用者只需输入实验的参数，如温度、注射次数、注射量等，计算机就可以完成整个实验，再由Origin软件分析ITC得到的数据)。测量时不需要制成透明清澈的溶液, 而且量热实验完毕的样品未遭破坏，还可以进行后续生化分析。尽管微量热法缺乏特异性但由于生物体系本身具有特异性，因此这种非特异性方法有时可以得到用特异方法得不到的结果，这有助于发现新现象和新规律，特别适应于研究生物体系中的各种特异

10、过程。ITC的关键优势之一是创建生物相关实验的独特能力。再没有其他技术能提供完全无标记且液相的分析环境，同时无需靶点高分子或配体的固定。ITC的应用在相关模型生物系统的建立和验证中起了重要的作用。测温滴定法以热效应为基础,与溶液的许多性质(如粘度、光学透明度、介电常数、溶剂强度、光谱性质和电学性质以及离子强度等)无关,因此可以用于气相、液相、非水溶液、有色溶液、胶体溶液和粘稠浆状等体系。2021/7/2615药剂学方面的应用we investigated the interactions between -CD and thetwo guest molecules by means of is

11、othermal titration calorimetry(ITC)2021/7/2616The sample cell was loaded with aqueous solutions of mPEG5k-ad (0.5 mM) and mPEG5k-chol (0.1 mM), respectively. Titrations were carried out by stepwise injection of -CD solutions (5 mM) into the sample cell (5 ml per injection, 60 s interval). All measur

12、ements were done at 25 0.1 C and 37 0.1 C.2021/7/26172021/7/2618The inflection points, which reflect the stoichiometry of the formed complexes, are close to one in case of mPEG5k-ad indicating that each adamantane group is associated with one -CD molecule. This is in agreement with other reports sho

13、wing a 1:1 binding stoichiometry between adamantane derivatives and -CD . In case of mPEG5k-chol, the stoichiometry was close to three, which had also been reported previously .The higher order of complexation may be due to self-association of cholesterol and/or partial inclusion of the cholesterol side chain into the -CD cavity We found that mPEG5k-chol exhibited a higher binding affinity than mPEG5k-ad2021/7/2619Applications include:Characterization of molecular interactions of small molecules.表征小分子的相互作用Lead optimization.先导物优化E