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等温滴定量热法的实验操作

Video Article

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Michael R. Duff,1, Jordan Grubbs1, Elizabeth E. Howell1

1Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee

Correspondence to: Elizabeth E. Howell at lzh@https://www.wendangku.net/doc/5115821068.html,

URL: https://www.wendangku.net/doc/5115821068.html,/video/2796

DOI: doi:10.3791/2796

Keywords: Molecular Biology, Issue 55, Isothermal titration calorimetry, thermodynamics, binding affinity, enthalpy, entropy, free energy

Date Published: 9/7/2011

Citation: Duff,, M.R., Grubbs, J., Howell, E.E. Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity. J. Vis. Exp. (55), e2796, doi:10.3791/2796 (2011).

Abstract

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment.

When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

Video Link

The video component of this article can be found at https://www.wendangku.net/doc/5115821068.html,/video/2796/

Protocol

Isothermal titration calorimetry (ITC) is a well established technique that can determine all the thermodynamic parameters (affinity, enthalpy and stoichiometry) of a binding interaction in one experiment.1 ITC works by titrating one reactant into a second reactant under isothermal conditions.

The signal measured is the heat released or absorbed upon interaction (binding) of the two reactants. A series of injections are performed and the heat signal will approach zero as the limiting reactant becomes saturated. Fitting of the isotherm gives the thermodynamic parameters.

Several reviews are available that describe the instrumentation as well as the math of data collection and analysis. 2,3 While other calorimeters are available (most notably the ITC200 with small volumes), here we describe a general protocol for the VP-ITC manufactured by MicroCal (now part of GE Healthcare).

1. Preparing Samples

1.In order to prepare the macromolecule and ligand in buffer, some potential issues need to be addressed. Accurate fits require proper

concentrations of the macromolecule, the species that typically goes in the sample cell of the ITC, and ligand, the species in the injection syringe. Because some proteins aggregate at the high concentrations needed for the species in the injection syringe, most often the protein is loaded into the sample cell. The optimal macromolecule concentration is determined from "c", the product of the predicted affinity of the system, which can be estimated using orthogonal methods prior to using ITC, and the total macromolecule concentration, where c = K a*[M].

Optimal values of c range from 10-1000, 1 though it is possible to get accurate data for weak-binding systems under specific experimental conditions with c-values below the lower limit.4 Thus, the macromolecule concentration must be determined with this range of c values in mind (i.e., for a K a of 106 M-1, macromolecule concentrations of 10 to 1000 μM should be used). Prior knowledge of the binding affinity of the system can help minimize the protein used for ITC through better design of the ITC experiment. The concentration of the ligand should be large enough (7-25 fold more concentrated than the K d for the weakest ligand binding site) so that saturation occurs within the first third to half of the titration. Accurate fitting of the data also requires saturation of the signal. For systems with higher binding affinity, a lower ligand concentration should be used to avoid saturation too early in the titration, which will give inaccurate fits. Once the heat of dilution control (i.e.

titration of ligand into buffer) has been subtracted from the titration, the enthalpy at saturation should approach zero.

2.Because small molecule impurities can give rise to artifactual signals in the ITC measurements, it is best, if possible, that the macromolecule

and ligand be exhaustively dialyzed against buffer. Alternatively, column chromatography, desalting spin columns, or buffer exchange

centrifugal filters (for example, Centricons) can be used to change the buffer of the macromolecule. If the ligand is a small molecule, it can be prepared using the dialysis buffer after the macromolecule has been dialyzed or by dialyzing against dialysis membranes with cutoffs suitable for small molecules (i.e. 100-500 Da for a Spectra/Por Float-A-Lyzer). Differences in the buffer composition between the ligand and macromolecule solutions can lead to signal artifacts from the heat of the dilution of impurities in the samples. After preparation, check to make sure the pH of the buffer, macromolecule and ligand match (± 0.05 pH units) as artifacts in the enthalpy can arise due to buffer protonation effects.5 Make sure to prepare enough of each species. For triplicate experiments and a dilution control, the amount of material needed will depend upon the ITC used. But, for most ITC instruments that have approximate 2 ml volume sample cells, at least 6-7 ml of

macromolecule solution will be needed, and can be conveniently prepared in 15 ml falcon tubes. For 300 μL injection syringes, 1-2 ml of solution should be adequate, and can be prepared in either falcon tubes or microcentrifuge tubes.

3.Dust, and other particulates, can cause artifacts in the baseline of the ITC thermogram. It is imperative that they be removed prior to running

the experiment. After preparation of sample stock solutions, the samples should be centrifuged in microcentrifuge tubes for five minutes at 8000 to 14,000 RPM to pellet particles in the solution. Remove the supernatant, being careful not to disturb the pellet, and place it in a new falcon/microcentrifuge tube.

If reductants are needed to maintain reduced cysteines, use low concentrations and fresh stocks of β-mercaptoethanol,TCEP (tris(2-

carboxyl)phosphine) or dithiothreitol to minimize any artifacts due to reductant oxidation. Also, the presence of organic solvents in the

buffer (common for some small molecule ligands that may need methanol or DMSO in order to be soluble) can cause signal artifacts. If organic solvents are needed for the ligand, then the macromolecule solution must also contain the same concentration to avoid any signal arising from the heat of dilution of the organic solvent. Slight differences in buffer composition between the ligand and macromolecule due to cosolvents, salts or pH are possible during preparation of the samples. It is best to check the dilution of the ligand into the sample buffer or the sample buffer into the macromolecule to ensure that heat signals arising from differences in the buffer content don't cause data arising solely from artifacts.

4.Check the concentrations of the macromolecule and ligand carefully using techniques suitable to your system (such as absorbance

measurements, HPLC ,colorimetric assays, BCA assays for proteins, etc) to record their exact concentrations. Differences in the actual concentration and the concentration used to fit the isotherm will cause errors in the stoichiometry, enthalpy and binding affinity determined from the experiment. It is common to degas the samples to avoid signal artifacts due to air bubbles or release of dissolved gases during the titration, particularly at higher temperatures.

2. Setting up the Experiment

1.Make sure the sample cell and injection syringe are cleaned according to the manufacturer's protocol prior to loading the macromolecule and

ligand. Rinse the sample cell two or three times with 1.8 ml of distilled water using the Hamilton syringe (use care with the Hamilton syringe as the barrel is easily broken or the needle bent). Next rinse the sample cell several times with 1.8 ml of buffer. Load the sample cell with 1.8 ml of macromolecule solution, being careful to avoid bubble formation.

2.Fill the reference cell with distilled water. For most buffers, distilled water is fine to use as the reference solution. However, for buffers with

particularly high ionic strengths or osmolality, it is better to use the buffer as a reference.

3.Remove air bubbles from the reference and sample cells using the Hamilton syringe. Gently move the needle of the syringe up-and-down the

sides of the cell knocking any bubbles that may be at the bottom of the cell and attached to the well to the top of the cell. Remove any excess volume from the sample and reference cells.

4.Attach a plastic syringe to the fill port of the injection syringe using tubing. Rinse the injection syringe with distilled water. Follow by a buffer

rinse. Make sure the injection syringe is completely evacuated by drawing air through the system. Place the needle of the injection syringe into the ligand solution and draw the ligand solution into the injection syringe until the entire syringe is full. Continue drawing a little excess volume (approximately 50 μl or more) into the port and attached tubing. Immediately close the fill port of the syringe and detach the tubing and plastic syringe. Purge and refill the injection syringe two more times to remove any bubbles from the syringe. Remove the syringe from the ligand solution and wipe the side with a kimwipe to remove any drops, being careful not to touch the syringe tip to the kimwipe as this may remove volume from the syringe. Also, be careful not to knock or jar the syringe as this also can cause loss of volume from the syringe tip.

Place the injection syringe into the sample cell.

5.Set up the parameters for running the ITC. For binding systems with strong heat signals, a large number of low volume injections will give

more data points for fitting (e.g. 75 injections of 3 μl). For systems that have weak heat signals, a small number of large volume injections are preferable (e.g. 33 injections of 8 μl). Most commonly, fewer injections with higher injection volumes are used. It may take several

titrations to optimize the conditions that are best for your system. It is important to note that the binding enthalpy can either be exothermic or endothermic, depending upon the system being studied. Unfortunately, some systems have low heat signals, making the heat of reaction difficult to determine. Problems with such systems can potentially be overcome by increasing the concentration of the macromolecule, changing the temperature of the experiment (depending upon the heat capacity of the binding system), and/or altering the pH or ionic

strength of the buffer.

6.Also consider the time spacing between each injection. It is imperative that after each injection of ligand, the system is given time to

equilibrate and the heat signal returns to baseline before the next injection occurs. For most systems, three to five minutes should be

adequate. The time between injections should be increased for systems where the equilibration doesn't occur within five minutes.

7.Because there will be some mixing between the macromolecule and ligand solutions in the injection syringe once it is inserted into the sample

cell, the first injection will give spurious results. It is best to use small volumes (e.g. 2 μl) for the first one or two injections (so they can later be discarded) and keep the subsequent injections at the desired values.

8.Choose the temperature of the experiment (with 25 °C being the most common, though temperatures between 2 and 80 °C can be used). It

is best to choose a temperature that matches other experiments (binding, kinetics, etc.) performed on the ligand-macromolecule system. The ITC can be set up to equilibrate at a temperature different from the temperature of the experiment. If the experiment is going to be performed at a temperature more than 10 °C away from room temperature, then it is best to set the equilibration temperature for the instrument within 5°C of the experimental temperature. This will decrease the time the instrument will take to reach the temperature of the experiment.

The stirring speed of the syringe also needs to be considered. Stirring is necessary for adequate mixing of the ligand and macromolecule during the titration, but some proteins are destabilized by rapid stirring. For these cases, the stirring speed should be set at a relatively low rate. Once all the experimental parameters have been set up, then the experiment can be started.

9.Once the experiment has finished, the ITC can be cleaned according to the manufacturer's protocol. The solution in the sample cell at the end

of the experiment can be kept if additional experiments on the macromolecule-ligand complex mixture are desired.

10.Repeat the titrations at least one or two more times to get reproducible data. Run a control where ligand is titrated into buffer in the sample

cell to determine the heat of dilution for the ligand. For some systems where there is cooperative binding, additional information about

the binding process can be gained by injecting the macromolecule into the ligand. The different injection orientations can give additional information, which may be helpful for global fitting.6

3. Analyzing the Data

1.Fitting of the data can be easily performed using macros in any data fitting program (usually supplied by the manufacturer along with the

instrument). Load the first data file. Check the raw thermogram for any signs of air bubbles or other artifacts in the signal. If there are any artifacts (such as spikes in the baseline or peaks when there was no injection at that time point), then note these data points as they should be removed. Next, load the ligand dilution control data, and subtract the ligand dilution data from the binding isotherm. At this time, remove all spurious data points, including the first one or two data points of the titration where dilution artifacts occur (refer to step 7).

2.Select the data fitting model (one binding site, two/multiple binding sites, cooperative binding, etc) to be used to fit the data. The data can

be fit with initial guesses of the fitting parameters, stoichiometry (n), enthalpy (ΔH) and binding affinity (K a). If prior knowledge of the binding affinity, or other parameters, is known from orthogonal experiments, then these values can be entered. This helps avoid the fit becoming trapped in local minima during fitting. Once the data have been fit for the first titration, repeat steps 15 and 16 for the rest of the isotherms.

Alternatively, the data can be fit globally using programs such as SEDPHAT.6 SEDPHAT may be particularly helpful in global fitting of binary complex datasets (i.e. molecules A+B) in combination with ternary complex datasets (molecules A+B+C). For example, in binding of molecule

C to an A-B complex, it is not always clear whether there might be any signal due to binding of C to A or of B to A (if A is not fully saturated).

3.To ensure that the ITC data are not artifactual, the binding affinity and stoichiometries obtained from ITC should be compared with an

orthogonal method.7,8 Additionally, the ITC enthalpy value can be compared to the enthalpy from a van't Hoff plot.9 It can also prove helpful to use different concentrations of the ligand or macromolecule as the absolute value of the heat signal should increase with increasing macromolecule concentration. Artifacts are most likely to arise if the buffers in the cell and syringe are not matched. Another possibility for artifacts arises from impure samples.

4.We recommend that the user start with simple binary complex titrations for which there is previous information available on K d and

stoichiometry. Then, if additional ligands bind, the user can try more complicated ternary complex titrations, varying the ligand concentrations, and perhaps switching the syringe and cell components. A comparison of enthalpies for both paths to ternary complex formation should be additive as enthalpy is a state function.10 Again, SEDPHAT6 is likely to be quite useful here.

4. Representative Results:

Figure 1. A representative example of a well-behaved titration for the binding of the cofactor NADPH to E. coli chromosomal dihydrofolate reductase (ecDHFR). Panel (A) shows the raw thermogram; (B), the binding isotherm from the integrated thermogram fit using the one-site model in the Origin software; and (C) the fit of the isotherm using the one binding site model from SEDPHAT along with fit residuals. From the Origin software, using the one site binding model, n = 1.09 ± 0.02, K d = 0.194 ± 0.001 μM, ΔH = -22.7 ± 0.4 kcal/mol, TΔS = -13.1 ± 0.4 kcal/mol, and ΔG = -9.16 ± 0.01 kcal/mol. Fits of the data using Sedphat afford an n of 0.94 ± 0.01, a K d of 0.195 ± 0.013 ΔM, a ΔH of -22.5 ± 0.2 kcal/mol, TΔS of -13.39 kcal / mol and ΔG of -9.15 kcal/mol.

Discussion

ITC has been used extensively in studying ligand macromolecule interactions,11 with studies looking at protein-ligand,12 DNA-ligand13 and RNA-macromolecule14 studies. ITC can even be run with solid materials, such as nanoparticles, that form uniform suspensions.15 Further, ternary systems, where one ligand is already bound to the macromolecule and a second ligand is titrated, can be used to determine the thermodynamics of, for example, binding of substrate to an enzyme-cofactor system.7 Studies can also be performed for molecules with very high binding affinities that would normally exceed the detection limit of the ITC by performing competition binding assays with weaker binding ligands.16 Information on weakly binding ligands can also be obtained by competition assays.17 The role of water in binding can be explored by ITC,18 along with the dependence of the enthalpy on solvent reorganization.19 Recently, the thermodynamics of conformational change of DNAK variants were measured upon binding of ADP and ATP.20 Protein-protein association can be studied by ITC, yielding information on hetero complexes21 as well as on homo-association.22 Temperature effects on the binding enthalpy will give the heat capacity of the binding event.2 The number of protons absorbed or released upon binding can also be determined from experiments performed in buffers with different enthalpies of ionization.5 If additional ITC studies are done at different pH values, then the pKa of the group involved can potentially be determined. 23

To summarize, accurate measurements of the concentration of the macromolecule and ligand are imperative for a good ITC experiment. Sample concentrations also need to be within a proper range to get reliable data. Care should be taken with the buffer as small molecule impurities and pH mismatches will cause artifacts in the thermogram.

Disclosures

No conflicts of interest declared.

Acknowledgements

This work was supported by NSF grant MCB-0817827.

References

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7.Bradrick, T.D., Beechem, J.M., & Howell, E.E. Unusual binding stoichiometries and cooperativity are observed during binary and ternary

complex formation in the single active pore of R67 dihydrofolate reductase, a D2 symmetric protein. Biochemistry. 35, 11414-11424 (1996).

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System. Biochemistry. 49, 4036-4042 (2010).

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Recognit. 23, 395-413 (2010).

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15.De, M., You, C.C., Srivastava, S., & Rotello, V.M. Biomimetic interactions of proteins with functionalized nanoparticles: a thermodynamic

study. J. Am. Chem. Soc. 129, 10747-10753 (2007).

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186-191 (2006).

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等温滴定量热法

等温滴定量热法(ITC)的简易操作流程： ①样品的准备，包括滴定物与被滴定物(如DNA滴定蛋白质)。 a.实验前的蛋白质样品需用缓冲溶液透析(注意透析袋的正确使用)，透析时间一般为24小时，buffer体积为1L，且中途注意更换buffer，其目的是为了减少由于溶液组成不同而产生的滴定误差； b.样品的浓度要求，一般要求的浓度为微摩尔级，且滴定物(DNA) 的浓度是被滴定物(蛋白质)的十倍左右为宜，且实验前需要再次确认所配样品的浓度是否符合要求； c.在进行滴定实验前，用于空白对照的缓冲液，蛋白样品以及 DNA均需抽真空除气泡(15Mins左右为宜)。 ②仪器的清洗。 a.在进行真空除气泡前，除气泡用容器均需用超纯水清洗三次左右，且清洗完毕后擦干内壁，防止由于残留缓冲液的稀释而导致样品浓度的改变； b.样品池(sample cell)的清洗，用超纯水清洗15次左右(每次2ml 左右)，清洗完毕后，一定要将残留的超纯水吸干净； c.注射器(syringe)的清洗，用超纯水清洗3次以上(专用注射器清洗,此时无需点击open,close和purge选项)，清洗完毕后将注射器的头部擦干，之后清洗装DNA的小试管，步骤同注射器的清洗。 ③设置空白对照试验（DNA滴定缓冲液），将缓冲液置于小试管中(为了防止空气的进入，一般添加样品的量大于其实际所需的量)，打开控制界面，点击open之后，用手动注射器缓慢拉动活塞，此时注射器管中的液面上升，然后点击close, 注射器会自动将小试管中的缓冲液吸入注射器中，当缓冲液完全吸入注射器之后点击pump键，除去气泡；在清洗样品池的同时就设置所需的实验温度（套管温度，一般较反应温度稍低），并输入样品的实际浓度以及实验数据文件名和储存路径等一系列参数（如注射时间，注射次数，注射间隔时间）。

等温滴定量热法应用

Example 2：Isothermal Titration Calorimetry for AIDS Drug Development 艾滋病药物的等温滴定量热法人们付出了大量努力，试图利用药物帮助艾滋病受害者减少艾滋病流行所造成病毒感染。热力学通过热力学解释实验热-滴定数据为此作出了贡献。如图2-1所示，在艾滋病毒感染人体细胞后，产生一系列艾滋病毒的复制步骤。受感染的细胞表达蛋白和蛋白酶；蛋白酶的作用在于蛋白酶切割聚蛋白，裂解的蛋白重新组装得到一个新的艾滋病病毒。图2-1 HIV蛋白酶在病毒复制过程中合成新的病毒为了防止形成新的病毒，可通过引入药物使使HIV蛋白酶失活。这种药物叫做蛋白酶抑制剂，可阻止多聚蛋白的分裂，如图2 – 2所示。图２-２通过一直HIV蛋白酶从而组织新病毒的生成蛋白酶和抑制剂的关系可以用传统的锁钥机制描述，如图2 – 3所示。抑制剂必须有正确的形状才能进入艾滋病毒蛋白酶的活性位点，其中，抑制剂是“钥匙”，必须保证适合蛋白酶这把“锁”。

然而，因为突变，艾滋病毒蛋白酶的活性位点可以以不同的形式存在，如图2 - 3所示；原株的活性位（锁）用“十”字表示，突变株活性位点（锁）用六边形表示。我们需要寻找一种同时适合这两种形状的“锁”的药物（钥匙），不仅可以和原株蛋白酶的活性位点结合也与突变株蛋白酶的活性位点结合。热力学可以帮助识别最佳候选药物。图２-３传统“锁-匙”机制图2-4列出两种候选药物1和2。药物2比1具有更好的适应性，同药物1相比的，它具有不对称的功能；甲苯基团的称性比叔丁基弱。此外，药物2更灵活，因为它有两个可旋转的键，而药物1只有一个。不对称和灵活性为药物提供了额外的构象，可以适应一个艾滋病毒突变位点。图２-４两种候选药物１和２为定量衡量药物的效果，Ｏhtaka和Freire利用等温滴定量热（ITC）的热力学分析数据得到所需结果。用A表示艾滋病毒蛋白酶，B表示抑制剂（药品）。我们定义一个解离常数Kd和它的倒数，缔合常数Ka，下标d表示分离，ａ表示缔合。其中，[]代表在水溶液中物质的浓度。对于好的药物,我们希望Kd很小或者Ka很大。在A + B ----AB的反应中，A(蛋白酶)和B(抑制剂)的结合由标准焓和标准熵决定。上o标表示标准状态。

等温滴定量热法的实验操作

Video Article Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity Michael R. Duff,1, Jordan Grubbs1, Elizabeth E. Howell1 1Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee Correspondence to: Elizabeth E. Howell at lzh@https://www.wendangku.net/doc/5115821068.html, URL: https://www.wendangku.net/doc/5115821068.html,/video/2796 DOI: doi:10.3791/2796 Keywords: Molecular Biology, Issue 55, Isothermal titration calorimetry, thermodynamics, binding affinity, enthalpy, entropy, free energy Date Published: 9/7/2011 Citation: Duff,, M.R., Grubbs, J., Howell, E.E. Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity. J. Vis. Exp. (55), e2796, doi:10.3791/2796 (2011). Abstract Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given. Video Link The video component of this article can be found at https://www.wendangku.net/doc/5115821068.html,/video/2796/ Protocol Isothermal titration calorimetry (ITC) is a well established technique that can determine all the thermodynamic parameters (affinity, enthalpy and stoichiometry) of a binding interaction in one experiment.1 ITC works by titrating one reactant into a second reactant under isothermal conditions. The signal measured is the heat released or absorbed upon interaction (binding) of the two reactants. A series of injections are performed and the heat signal will approach zero as the limiting reactant becomes saturated. Fitting of the isotherm gives the thermodynamic parameters. Several reviews are available that describe the instrumentation as well as the math of data collection and analysis. 2,3 While other calorimeters are available (most notably the ITC200 with small volumes), here we describe a general protocol for the VP-ITC manufactured by MicroCal (now part of GE Healthcare). 1. Preparing Samples 1.In order to prepare the macromolecule and ligand in buffer, some potential issues need to be addressed. Accurate fits require proper concentrations of the macromolecule, the species that typically goes in the sample cell of the ITC, and ligand, the species in the injection syringe. Because some proteins aggregate at the high concentrations needed for the species in the injection syringe, most often the protein is loaded into the sample cell. The optimal macromolecule concentration is determined from "c", the product of the predicted affinity of the system, which can be estimated using orthogonal methods prior to using ITC, and the total macromolecule concentration, where c = K a*[M]. Optimal values of c range from 10-1000, 1 though it is possible to get accurate data for weak-binding systems under specific experimental conditions with c-values below the lower limit.4 Thus, the macromolecule concentration must be determined with this range of c values in mind (i.e., for a K a of 106 M-1, macromolecule concentrations of 10 to 1000 μM should be used). Prior knowledge of the binding affinity of the system can help minimize the protein used for ITC through better design of the ITC experiment. The concentration of the ligand should be large enough (7-25 fold more concentrated than the K d for the weakest ligand binding site) so that saturation occurs within the first third to half of the titration. Accurate fitting of the data also requires saturation of the signal. For systems with higher binding affinity, a lower ligand concentration should be used to avoid saturation too early in the titration, which will give inaccurate fits. Once the heat of dilution control (i.e. titration of ligand into buffer) has been subtracted from the titration, the enthalpy at saturation should approach zero. 2.Because small molecule impurities can give rise to artifactual signals in the ITC measurements, it is best, if possible, that the macromolecule and ligand be exhaustively dialyzed against buffer. Alternatively, column chromatography, desalting spin columns, or buffer exchange centrifugal filters (for example, Centricons) can be used to change the buffer of the macromolecule. If the ligand is a small molecule, it can be prepared using the dialysis buffer after the macromolecule has been dialyzed or by dialyzing against dialysis membranes with cutoffs suitable for small molecules (i.e. 100-500 Da for a Spectra/Por Float-A-Lyzer). Differences in the buffer composition between the ligand and macromolecule solutions can lead to signal artifacts from the heat of the dilution of impurities in the samples. After preparation, check to make sure the pH of the buffer, macromolecule and ligand match (± 0.05 pH units) as artifacts in the enthalpy can arise due to buffer protonation effects.5 Make sure to prepare enough of each species. For triplicate experiments and a dilution control, the amount of material needed will depend upon the ITC used. But, for most ITC instruments that have approximate 2 ml volume sample cells, at least 6-7 ml of

结构化学实验-itc等温量热滴定

一、实验目的： 1.了解MicroCal iTC200等温滴定量热仪在测量蛋白质相互作用中的应用 2.了解仪器基本工作原理，学习蛋白质相互作用的测定步骤和仪器操作 3.简要分析实验结果。二、实验原理：在研究两种或两种以上的蛋白质的功能时，相关蛋白质之间常常存在相互作用（常常是氢键或范德华力），如果两蛋白可以彼此结合，则结合的过程中会放出一定的热量。所以，通过测定蛋白质相互作用时放出热量的大小，可以得到蛋白相互作用时的结合常数KD、化学计量比N和焓变ΔH，从而由热力学公式ΔG = RT lnKD和ΔG = ΔH -TΔS可以进一步得到反应的自由能变化。

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。 MicroCal iTC200等温滴定量热仪可以用来定量测定生物分子间的相互作用，例如蛋白质-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-细胞相互作用等。从而获得亲和力以及相关热力学数据。通过滴定操作和热量的测量，量热仪可以给出热量-摩尔比曲线：图像中曲线的突跃中点对应的化学计量比就是两种蛋白质相互作用的化学计量数N，突跃中点处曲线的斜率就是两种蛋白相互作用的结合常数KD。决定曲线形状的主要参数是C值： C = 滴定池中的蛋白浓度/ K D = [M]t/ KD × N

教育观察法及案例分析

教育观察法及案例分析观察法是由研究者经过自己的感官等方式搜集资料。教育观察法虽然也通过研究者的亲身感受或体验来获得研究对象的感性材料，即以日常观察为基础，但它不是自发的、偶然性的活动，而是有目的、有计划的活动，观察对象与方法也是经过选择与策划的；并且最后需要作严格详细的观察记录。相对于其他教育科研方法来说，观察法收集第一手感性材料的必经途径，是后继研究的基础和起点，同时可以检验科学假说、发展科学理论。一般来说，观察法的实施有以下要求： 1、明确观察的具体目标与要求 2、准备好观察手段 3、进行多次反复观察 4、客观、全面、典型、有计划地进行观察 5、要掌握好观察方法与技术 6、及时分析、处理观察所得的材料其实施步骤包括：一、准备工作 1、制定观察计划与提纲观察提纲包括：谁、什么、何时、何地、如何、为什么(who, what, when ,where, how, why) 2、准备观察所用的辅助工具，记录表格、记录方式、仪器设备 3、确定观察途径：访谈、听课、参观、参与 4、训练观察人员二、实施观察先熟悉环境，再建立和谐关系，最后正式观察。注意做到灵活地执行计划，抓住观察重点，注意做到看、听、问、查、思考相结合，做好观察记录三、观察资料的整理与分析，撰写研究报告在本次作业中，我选取了三个教育观察法案例，分别是《大班幼儿与一年级小学生24小时活动观察比较》，《幼儿个体行为观察》和《优化情境教学”课堂观察分析》，有针对性的进行分析。

案例1——大班幼儿与一年级小学生24小时活动观察比较课题名称：大班幼儿与一年级小学生24小时活动观察比较研究研究人员：杭州天长小学教师——成游李任斌协作人员：与幼儿、小学生有关的教师、家长配合。前言：幼儿园与小学的衔接是一个长期没有解决的问题。幼儿园跟小学的衔接涉及到教学内容、教学方法、学习习惯、作息制度等许多问题。根据小学低年级老师和家长反映，学生入学后学习负担和思想负担较重，健康状况下降，对小学学习生活不习惯的现象十分明显，儿童入学前后活动安排上的差别已成为幼儿园与小学衔接上急待研究解决的问题之一。我们拟通过对幼儿园大班幼儿与小学一年级学生在24小时内各类活动时间的调查获得数量资料，为幼儿园与小学儿童活动安排的衔接问题提供依据。研究对象：在本区九所幼儿园大班幼儿中随机选择50余名幼儿，并从本区五所小学中，选择50余名去年从这九所幼儿园中毕业、现在小学一年级学习的小学生。与家长联系，排除一小部分由于其他原因使家长无法配合观察的对象。原则上定为幼儿与小学生各40—50名。研究方法：（1）观察内容为一天24小时内的全部活动内容。（2）儿童在幼儿园或小学内的活动情况由教师跟踪观察并作详细记录，在园外、校外活动情况由家长进行观察记录。（记录草表附后）（3）观察日期不统一定为哪一天，但是，对每一个儿童的观察，教师与家长，应在同一天进行。观察日子应排除星期天、幼儿园或小学组织半天以上校外活动的日子，小学还须排除周六。（因为那天下午不上课）（4）为了熟悉观察分类与要求，并形成及时记录的习惯，要求观察者特别是家长在正式观察前三天之内至少应试观察一小时以上。儿童活动名称及归类标准：

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申明：本资料来源于网络，版权归原作者所有！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特优势，样品用量小，方法灵敏度和精确度高(本仪器最小可检测热功率2 nW，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

观察法案例及分析

优缺点：时间取样法可以全面了解行为或事件发生的过程，节省收集资料的时间，适用性也比较广；但是由于观察者注重行为发生时的状况，可能对整个情境缺乏完整的了解。对于我们将要走上教师岗位的人来说，事件记录法有重要的作用，可以用来对班级某种现象或是某个学生的特定行为进行观察记录，从而更好地了解他们。案例三：陈鹤琴对儿子的观察——日记描述法幼儿教育家陈鹤琴用日记方式记录了儿子陈一鸣自出生起来的发展，观察了808天，发表了《儿童心理之研究》一书。日记节选：第一天 1.这个孩子是1920年12月26日凌晨2点零9分生的。 2.生后两秒就开始大哭，一直哭到2点19分，共连续哭了10分钟。 3.出生后45分钟就打哈欠。 4.出生后2点44分钟，又开始打哈欠，以后再打哈欠6次。 5.出生后的12十点钟，生殖器开始能举起，这大概是因为膀胱盛满尿的缘故，随即就小便。 6.同时大便是一种灰黑色的流汁。 7.用手扇他的脸，他的皱眉肌就皱缩起来。 8.用指头触他的上唇，上唇就动。 9.打喷嚏两次 …… 分析：日记法是研究儿童行为较早的一种方法，需要在较长时间内反复观察幼儿的行为，持续地记录变化，记录新的发展和新的行为。优缺点：作为个案研究的记录可以对幼儿进行系统完整的纵向分析，并能发现一些儿童成长的规律；但是日记描述法取样有限不具有代表性，而且耗费时间和精力，效率比较低。新发展：日记记录法的理念可以借助日新月异的技术手段来更好地实现，如用

等温滴定量热法在生命科学研究中应用

等温滴定量热法在生命科学研究中应用等温滴定量热法(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特优势，样品用量小，方法灵敏度和精确度高(本仪器最小可检测热功率2 nW，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/RN A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 微量热技术(Microcalorimetry)(2008-05-27 19:58:14)

等温滴定量热仪和差示扫描量热仪在生物制剂研发中的应用

等温滴定量热仪和差示扫描量热仪在生物制剂研发中的应用液剂中蛋白药物的稳定的一个重要因素是适当的辅料的选择，适宜的辅料浓度能够在延长药物保质期的同时确保患者的最高的用药安全。尽管过去十年间的文献报道了维持稳定性的一般准则，但是在存储期间辅料如何提升蛋白药物的稳定性，其机制尚未完全明晰。为能合理优化蛋白制剂，掌握有关蛋白-辅料相互作用机理的知识十分重要。通过微量热技术来研究来探索蛋白-辅料的相互作用，因而被越来越多的应用于生物制剂的研发和优化。通过使用等温滴定量热仪（ITC）和差示扫描量热仪（DSC）等技术，评估与相互作用有关的热力学参数，例如辅料-蛋白的结合，辅料参与下蛋白质伸展等，揭示研发最优制剂所需的重要机理信息。这里我们列举两个ITC和DSC是怎样辅助蛋白制剂研发的例子。在第一个例子中，使用ITC揭示了聚山梨酯80（常用表面活性剂，通过降低蛋白表面吸附和聚集以稳定蛋白性质）与蛋白X（ProX）之间的相互作用。使用单位点模型拟合ITC数据计算得出结合亲和常数（K A）=1430±260 M-1，结合焓（△H）=-6.3±1.1 kcal/mol，每个ProX 分子结合位点数（n）=2.6±0.3。另外一个例子中，使用DSC和ITC产生的数据集显示在抗菌防腐剂，苯酚存在的条件下，ProX在pH5.7时结构最稳定。因而在上述两个研究中，来自于GE Healthcare生命科学部的等温滴定量热仪ITC200 和差示扫描量热仪VP-Capillary DSC在探寻制备ProX最佳制剂方法方面提供了非常重要的信息。由于蛋白质分子内在的不稳定性，包括物理不稳定性（伸展、聚集、吸附）和化学降解性（氧化、脱酰胺、断裂），以基于蛋白质的治疗发展面临着巨大的挑战。蛋白的不稳定可以导致蛋白活性降低甚至可能产生潜在的免疫原性。为增加蛋白的稳定性，可以尝试改变蛋白所在溶剂的性质，包括选择缓冲液系统，调节pH值，添加辅料/添加剂，即研发最佳剂型。选择适当的赋形剂并优化赋形剂浓度是蛋白药物的溶剂稳定性的一个重要因素，可以增加用药的安全性并延长保质期。因而蛋白药物制剂研发的一个重要环节是赋形剂的选择，需满足以下条件，合适的溶解度，无毒，可维持蛋白结构的完整，保证较长的保质期，以及保持产品的生物活性。这样的辅料包括氨基酸、盐、金属、表面活性剂、糖，多羟基化合物和聚合物。可作为稳定剂，表面活性剂，抗菌防腐剂或抗氧化剂。赋形剂的稳定作用通常是蛋白特异性并呈现浓度依赖性。

等温滴定量热法(ITC)

等温滴定量热技术摘要：生物大分子可以和很多配体特异性结合，当物质结合时，热量要么产生，要么吸收。生物大分子与配体相互作用的定量描述需要确定反应过程中热力学参数的变化。相互作用过程中产生的热量变化可以用量热计定量监测。等温滴定量热技术（Isothermal Titration Calorimetry, 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，它已经成为鉴定生物分子间相互作用的首选方法。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，如结合常数（Ka）、结合位点数（n），结合焓（△H）、熵（△S）、恒压热容（△Cp）和动力学数据（如酶促反应的Km和kcat ）。这些信息提供了生物分子相互作用的真实写照。由于几乎所有的生化反应过程都有热量变化，所以ITC具有很广泛的应用，它可以应用于蛋白质-蛋白质相互作用、蛋白质折叠/去折叠、蛋白质-小分子相互作用、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-细胞相互作用等方面。关键字：等温滴定量热技术、相互作用、热力学商业化的测量生物分子相互作用热量的灵敏的量热计出现在上世纪80年代后期[1]。从此这种技术被广泛应用。在过去的20年中，等温滴定量热技术（ITC）成为研究相互作用的常用方法。随着现代ITC仪器的发展，ITC更加灵敏、快速、易用。分子识别是一个复杂的过程，是生命活动的基础。生物分子识别过程需要结合反应的热力学参数来阐明。等温滴定微量量热法可以直接定量检测滴定反应过程中的热量变化，确定反应的结合常数K B 、结合计量比（n）、反应焓变（?H）、熵变（?S）、恒压热容（△Cp）和动力学数据（如酶促反应的Km和kcat ）等热力学参数，用来表征生物分子间的相互作用。ITC已成为研究生物大分子结合反应的有力工具。现代的ITC仪器可以测量0.1μcal (0.4 μJ)的热效应，108–109M-1的结合常数，反应速率可以精确至10-12 mol/s，Km和kcat的测量范围分别在10-2–103mM 和0.05–500s[2]。由于几乎所有的生化反应过程都有热量变化，ITC 的应用非常广泛，包括涉及焓变的化学和生化结合反应以及一些复杂过程，比如酶动力学。与其他的研究方法相比，ITC 不需要作任何的化学修饰或固定，可以直接地测量结合焓。许多生化反应可以在等温条件下完成，通过滴定改变样品的组成，为ITC 提供了广阔的潜在应用前景。它对于阐明分子结合机制，结合分子结构进行药物设计等都有重要的作用。等温滴定量热技术简介 ITC是一种热量连续变化的量热器。主要由隔热夹套包裹着的样品池(反应池)和参比池、注射器和一台计算机组成，注射器同时具有搅拌作用，计算机控制温度控制装置和信息反馈系统（图1）[3]。恒温条件下当滴定进行前，首先反馈系统给样品池一个连续的能量确定基线。滴定进行时，注射器滴入反应物（通常是用小分子物质滴定大分子，本文中注射器中的反应物称作配体，ligand，L）与样品池中被滴定物（本文中被滴定的反应物称作大分子，macromolecule，M）相互作用触发结合反应，形成大分子/配体复合物（macromolecule/ligand，ML）。复合物的形成伴随着能量的释放或吸收，导致样品池温度的变化，参比池始终保持在实验温度，反馈系统提供热或降低热量来补偿样品池的温度变化，每注射一次后系统恢复至平衡状态再进行下一滴滴定。结果以峰的形式表示平衡温度偏移所需要的能量，峰的面积相

观察法的案例分析

观察法的案例分析最近几年，教学研究已不再在空洞的教学理论上进行研究，而是向学校回归，向教师回归，向课堂教学实践回归。越来越多的研究理论的工作者和教育实践者认为“学校应该成为教师专业发展的摇篮”，学校应该成为理论研究的基地。而最容易付之实践的教学方法，是观察的实践与研究。教师的观察是根据课题的需要而进行的观察，为解决某个问题而主动进行的观察，并在教学实践中得到直接的经验和事实素材。教师有了这些观察的对象后，就要进一步深刻分析、归纳总结、探寻规律，深入研究，并运用科学的方法，改变教学策略，进一步进行教学实践，以取得更好的教学效果。成佳肴是三年级（2）班的一名很可爱的小女孩，但她给所有老师留下的印象也就只有可爱而已，因为她学习起来反应很慢，让人感觉很着急，成绩上也不怎么样出色。刚带上她数学时，的确让我感觉挺头疼的，可后来我发现她的父母非常重视她的学习，和老师配合的也很好，环境对一个人的影响是相当大的，所以我相信成佳肴生活在这样的家庭中有朝一日肯定会很出色，只是需要足够的时间。于是我决定先从各方面去了解观察下成佳肴，通过一段时间的学习观察，我发现她是一个急性子的女孩，就是在学习上反应比较慢，可是她并不是不会的慢，只要慢慢来她能做到很高的准确率。根据她这一点每次做练习或考试前，我都会单独提醒一下她“不要着急，慢慢来，你并不是不会”。渐渐的我发现成佳肴不仅学习成绩上有了一定的提高，学习数学的兴趣和自信心也越来越高了，遇到不懂的题能及时来问，课上发言也很积极。由此看来观察法一般具体体现在以下几个方面：（1）观察法用于教师的课堂教学活动，有助于提高教学效率。（2）观察法用于观察学生的行为，了解学生，提高学生的学习效率。（3）观察法还用于促进

等温量热滴定法

滴定量热法也称热滴定法或测温滴定法。是一种量热或分析技术，即用一种反应物滴定另一种反应物，随着加入滴定剂的数量的变化，测量反应体系温度的变化。滴定一般在尽可能接近绝热的条件下进行，被滴定物可以是液体或悬浮的固体；滴定剂可以是液体或气体。温度变化是由滴定剂与被滴定物间的化学作用或物理作用（例如一种有机分子吸附于固体表面）引起的。实验数据以热谱图形式表示，它提供了有关反应中物质的量（滴定终点）和反应物质的特性（焓变）的数据。对图进行分析，可以得知反应容器中发生的反应的类型和数目，以及溶液中存在的各物种的浓度等信息。这部分内容称为热滴定，同时还可以确定反应的化学计量关系，计算反应的热力学量,如平衡常数K(ΔG°)、标准状态下的焓变ΔH°和熵变ΔS°,这部分内容称为滴定量热法。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、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。图1为滴定量热计框图。滴定管内含有一种反应物的滴定剂，可加入到反应容器内，滴定剂的浓度一般为被滴定液的数十倍甚至一百倍左右。反应产生的温度变化由温度传感器（例如热敏电阻）感知，通过惠斯通电桥电路转换为相应的伏特数或热功率。这个伏特数由放大电路放大并记录于记录仪上，或者馈入其他的数据采集系统。反应容器和滴定管置于同一恒温浴中，恒温浴温度由温度控制器控制，并由温度传感器测量。滴定剂和反应容器的温度必须相等，或者它们之间的温差必须精确知道。滴定剂的加入方式①连续加入法：滴定剂以恒速加入，可以得到滴定进程中热效应的完整记录。对于特别复杂的反应，可以从这种记录图中选择任意数目的数据点，用于必要的计算。连续滴定法要求对温度变化的响应必须够快，并且此法只适用于快速反应。②分次递增加入法：间歇地每次加入单位增量的滴定剂，在每次加入单位增量前，必须调节反应容器的温度，使回复到起始温度，通过加入许多次增量，也能够得到足够的测量数据，从而绘制成与连续法相似的图线。不过，它比连续法的工作量要大一些。递增法的好处是可以精确研究动力学上较慢的反应。③连续流动量焓法：使两种溶液各自以恒速连续流过一个混合(测量)容器。此法的优点是反应物于不存在气相情况下混合，这对于测量挥发性液体体系非常重要，并且可以忽略表面吸附效应。该法特别适用于研究复杂的生物化学和微生物学过程。此外，此法的装置可以连接在生产线中进行分析而达到过程控制的目的。

MicroCal等温滴定量热仪

MicroCal 等温滴定量热仪MicroCal iTC200 / Auto-iTC200 Imagination at Work

等温滴定量热 Isothermal Titration Calorimetry 等温滴定量热（ITC）是一种直接测量生物分子结合过程中的放热或者吸热的技术。通过对热量的测定可以精确的得到结合常数（K B），反应的化学计量数（n），熵（ΔS）和焓（ΔH），在单次实验中即可提供一整套有关分子相互作用的完整信息。ITC是表征生物分子相互作用的经典方法之一。 ITC的应用范围包括：小分子、蛋白、抗体、核酸、脂类及其他生物分子之间的相互作用特征鉴定；酶动力学；分析因分子结构改变导致的结合变化等。在全球，MicroCal 仪器被普遍应用于制药厂商、生物技术公司以及众多大学、科研院所和政府机构。工作原理 ITC可主要分为两个部分，上部是控制滴定的注射系统，下部是样品池和量热系统。注射器装载实验检测用的“配体”溶液，样品池中含有待检测的另一种“大分子”样品。实验时，在恒定的温度下，配体溶液以设定的体积和次数逐滴进入样品池中。两者发生相互作用时释放或吸收的热量和两者结合的数量之间存在间接比例关系。通过灵敏的热量检测系统和功率反馈补偿机制，便可准确的检测到反应过程中极其微小的的热量变化，并以实时的数据曲线形式输出。当样品池中的大分子逐渐被滴入的配体所饱和时，相互作用产生的热量信号会逐渐减小，直到最后仅能检测到配体滴入时产生的背景稀释热量。输出的曲线经过软件分析，便可得到全面完整的热动力学数据。图1.典型ITC数据图：上半部分表示每次注射采集的数据。下半部分表示每次注射的释放的热量（上图中每个峰的面积积分）对滴定样品与样品池中样品的摩尔比作图图2.ITC功率反馈补偿机制

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等温滴定量热法

等温滴定量热法应用

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等温滴定量热法(ITC)

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MicroCal等温滴定量热仪