等温滴定微量热仪(ITC)简介

等温滴定微量热仪(ITC)简介

等温滴定量热法在生命科学研究中应用

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等温滴定量热法(Isothermal Titration Calorimetry, ITC)是近年来发展起来的一种研究生物热力学与生物动力学的重要方法，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。微量热法具有许多独特之处。它对被研究体系的溶剂性质、光谱性质和电学性质等没有任何限制条件，即具有非特异性的独

最小可检测热效应0.125uJ，生物样品最小用量0.4ug，温度范围2 0C - 80 0C，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过程。

ITC的用途

获得生物分子相互作用的完整热力学参数，包括结合常数、结合位点数、摩尔结合焓、摩尔结合熵、摩尔恒压热容，和动力学参数(如酶活力、酶促反应米氏常数和酶转换数)。

ITC的应用范围

蛋白质-蛋白质相互作用(包括抗原-抗体相互作用和分子伴侣-底物相互作用)；蛋白质折叠/去折叠；蛋白质-小分子相互作用以及酶-抑制剂相互作用；酶促反应动力学；药物-DNA/RNA相互作用；RNA折叠；蛋白质-核酸相互作用；核酸-小分子相互作用；核酸-核酸相互作用；生物分子-细胞相互作用；……

加样体积：（实际体积）

cell：1.43 ml，syringe：300 μl

准备样品体积（最少量）

cell：2 ml，syringe：500 μl

样品浓度cell：几十μM到几mM

syringe：几百μM到几十mM

测量Kb范围102-1012 M-1

滴定实验前恒温30-60 min

等温滴定量热实验所需时间，一般1.5-4 hr

Sample Preparation Guidelines (ITC).

Proper sample preparation is essential for successful ITC testing. In particular, the minimal guidelines below must be strictly followed to insure an accurate estimate of stoichiometry (n), heat of binding (H), and binding constant (Kb) (or dissociation constant Kd = 1/Kb).

1.) The macromolecule solution (the sample to be placed in the reaction cell) must have a volume of at least

2.1 ml. The lowest concentration which can be studied is 3 M and this is adequate only for tight binding where Kd is smaller than 1 M. For weaker interactions, the macromolecule concentration should be 5 times Kd, or higher if possible. Preferably, the macromolecule solution should be dialyzed exhaustively against buffer for final equilibration.

2.) The ligand solution (the sample to be placed in the injection syringe) must have a volume of at least 0.7 ml. Its concentration should be at least 10 times higher than the concentration of macromolecule (if the macromolecule has multiple binding sites for ligand, then the ligand concentration must be increased accordingly). The buffer solution in which the ligand is dissolved should be exactly the same buffer against which the macromolecule has been equilibrated.

3.) After both solutions have been prepared, the pH of each should be checked carefully. If they are different by more that 0.05 pH units, then one of the solutions must be back-titrated so they are within the limit of 0.05 pH units. If any particles are visible in either solution, they should be filtered out.

4.) If possible, the concentrations of both solutions should be accurately determined after final preparation. Accurate determination of binding parameters is only possible if concentrations of binding components are known precisely.

5.) At least 20 ml of buffer must be sent along with the two samples, since this is used for rinsing the cell and for dilution if necessary.

6.) If possible, DTT should be avoided as a disulfide reagent and replaced by -mercaptoethanol or TCEP.