等溫滴定量熱法

1、等溫滴定微量熱法等溫滴定微量熱法(Isothermal Titration Calorimetry, ITC)林杰Page 22ITC Isothermal titration calorimetry (ITC) is used to measure the heat adsorbed or released during changes in the composition of a systemundergoing a titration process. 等溫滴定量熱技術(shù)（ITC）是一種監(jiān)測(cè)由結(jié)合成分的添加而起始的任 何化學(xué)反應(yīng)的熱力學(xué)技術(shù)，即用一種反應(yīng)物滴定另一種反應(yīng)物，隨著加入滴定劑

2、的數(shù)量的變化，測(cè)量反應(yīng)體系溫度的變化Page 33historybuilt 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 aromatic compou

3、nds 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靈敏度的提高靈敏度的提高Page 44historySince the beginning of 1990s, the number

4、of published papers related to isothermal titration calorimetry has symptomatically increased due to rapid diffusion of new commercial calorimeters in the scientific community.Page 55historyDuring the decade of 1990, isothermal titration calorimetry has evolved from a specialist method to a widely u

5、sed 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 .Page 66ITCPage 77在恒溫下，注射器中的“配體”溶液

6、滴定到包含“高分子”溶液的池中。當(dāng)配體注射到池中，兩種物質(zhì)相互作用，釋放或吸收的熱量與結(jié)合量成正比。當(dāng)池中的高分子被配體飽和時(shí)，熱量信號(hào)減弱，直到只觀察到稀釋的背景熱量。Page 88上圖：上圖：橫坐標(biāo)：時(shí)間橫坐標(biāo)：時(shí)間縱坐標(biāo)：熱功率縱坐標(biāo)：熱功率峰底與峰尖之間的峰面積為峰底與峰尖之間的峰面積為每次注射時(shí)釋放或吸收的總每次注射時(shí)釋放或吸收的總熱量。熱量。下圖：下圖：橫坐標(biāo)：滴定物與樣品溶液橫坐標(biāo)：滴定物與樣品溶液的摩爾比的摩爾比縱坐標(biāo)：滴定產(chǎn)生的總熱量縱坐標(biāo)：滴定產(chǎn)生的總熱量反應(yīng)過程的結(jié)合等溫曲線反應(yīng)過程的結(jié)合等溫曲線圖形Page 99ITC可以直接測(cè)量焓變H，結(jié)合常數(shù)Ka，而不對(duì)反應(yīng)體系產(chǎn)生

7、影響，也不引入修飾基團(tuán)，因此測(cè)得的結(jié)果更加可信典型的ITC數(shù)據(jù)配體溶液20次注射到ITC池的蛋白溶液中。每個(gè)注射峰（上圖）下方的區(qū)域與注射所釋放的總熱量相等。當(dāng)這種綜合的熱量相對(duì)添加到池中的配體摩爾比作圖時(shí)，就獲得了相互作用的完整結(jié)合等溫線（下圖）。用單位點(diǎn)模型來驗(yàn)證數(shù)據(jù)?；瘜W(xué)計(jì)量、結(jié)合常數(shù)及焓的數(shù)值都顯示在框內(nèi)。Page 1010流程流程1 1、確定合適的反應(yīng)物濃度、確定合適的反應(yīng)物濃度2 2、準(zhǔn)備樣品、準(zhǔn)備樣品3 3、滴定收集數(shù)據(jù)、滴定收集數(shù)據(jù)4 4、校正后的數(shù)據(jù)非線性回歸得到熱力學(xué)參、校正后的數(shù)據(jù)非線性回歸得到熱力學(xué)參數(shù)數(shù)5 5、分析模型、分析模型1、微克級(jí)、微克級(jí)3、配體濃度大于大分子

8、濃度、配體濃度大于大分子濃度1、考慮反應(yīng)物性質(zhì)、考慮反應(yīng)物性質(zhì)2、盡量避免稀釋熱、盡量避免稀釋熱3、抽氣處理、抽氣處理1、設(shè)置對(duì)照組、設(shè)置對(duì)照組2、平行試驗(yàn)、平行試驗(yàn)3、Origin軟件軟件Page 1111模型分析模型分析正確理解反應(yīng)過程首先要理解模型給出的各種參數(shù)的正確意義。根據(jù)模型給出的各種熱力學(xué)參數(shù)確定反應(yīng)的相關(guān)性質(zhì)。結(jié)合位點(diǎn)數(shù)不同，平衡常數(shù)的物料平衡的表達(dá)公式是不同的Page 1212Page 1313characteristics它通過高靈敏度、高自動(dòng)化的微量量熱儀連續(xù)、準(zhǔn)確地監(jiān)測(cè)和記錄一個(gè)變化過程的量熱曲線，原位、在線和無損傷地同時(shí)提供熱力學(xué)和動(dòng)力學(xué)信息，它已經(jīng)成為鑒定生物分子間

9、相互作用的首選方法?？色@得生物分子相互作用的完整熱力學(xué)參數(shù)，包括結(jié)合常數(shù)（Ka）、結(jié)合位點(diǎn)數(shù)（n）、摩爾結(jié)合焓（H）、摩爾結(jié)合熵（S）、摩爾恒壓熱容（Cp），和動(dòng)力學(xué)參數(shù)(如酶促反應(yīng)的Km和kcat）,用來表征生物分子間的相互作用。Page 1414獨(dú)特特點(diǎn)：獨(dú)特特點(diǎn)： 樣品用量小，方法靈敏度和精確度高(儀器最小可檢測(cè)熱功率2 nW，最小可檢測(cè)熱效應(yīng)0.125uJ，生物樣品最小用量0.4ug，溫度范圍2 - 80 ，滴定池體積(1.43 ml)。 實(shí)驗(yàn)時(shí)間較短(典型的ITC實(shí)驗(yàn)只需30-60分鐘，并加上幾分鐘的響應(yīng)時(shí)間)， 操作簡(jiǎn)單(整個(gè)實(shí)驗(yàn)由計(jì)算機(jī)控制，使用者只需輸入實(shí)驗(yàn)的參數(shù)，如溫度、注射

10、次數(shù)、注射量等，計(jì)算機(jī)就可以完成整個(gè)實(shí)驗(yàn)，再由Origin軟件分析ITC得到的數(shù)據(jù))。 測(cè)量時(shí)不需要制成透明清澈的溶液, 而且量熱實(shí)驗(yàn)完畢的樣品未遭破壞，還可以進(jìn)行后續(xù)生化分析。 盡管微量熱法缺乏特異性但由于生物體系本身具有特異性，因此這種非特異性方法有時(shí)可以得到用特異方法得不到的結(jié)果，這有助于發(fā)現(xiàn)新現(xiàn)象和新規(guī)律，特別適應(yīng)于研究生物體系中的各種特異過程。 ITC的關(guān)鍵優(yōu)勢(shì)之一是創(chuàng)建生物相關(guān)實(shí)驗(yàn)的獨(dú)特能力。再?zèng)]有其他技術(shù)能提供完全無標(biāo)記且液相的分析環(huán)境，同時(shí)無需靶點(diǎn)高分子或配體的固定。ITC的應(yīng)用在相關(guān)模型生物系統(tǒng)的建立和驗(yàn)證中起了重要的作用。 測(cè)溫滴定法以熱效應(yīng)為基礎(chǔ),與溶液的許多性質(zhì)(如粘度

11、、光學(xué)透明度、介電常數(shù)、溶劑強(qiáng)度、光譜性質(zhì)和電學(xué)性質(zhì)以及離子強(qiáng)度等)無關(guān),因此可以用于氣相、液相、非水溶液、有色溶液、膠體溶液和粘稠漿狀等體系。Page 1515藥劑學(xué)方面的應(yīng)用we investigated the interactions between -CD and thetwo guest molecules by means of isothermal titration calorimetry(ITC)Page 1616The sample cell was loaded with aqueous solutions of mPEG5k-ad (0.5 mM) and mPEG5

12、k-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 measurements were done at 25 0.1 C and 37 0.1 C.Page 1717Page 1818The inflection points, which reflect the stoichiometry of the forme

13、d 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 showing a 1:1 binding stoichiometry between adamantane derivatives and -CD . In case of mPEG5k-chol, the stoichiometry was close to th

14、ree, 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-adPage 1919Applications include:Characterization of molecular interactions of small molec