Acquisition of precise and accurate results by isothermal titration calorimetry (ITC) can be achieved through thoughtful experimental design and modeling and careful experimental operations. Large reported errors in ITC results in determinations of stoichiometries, equilibrium constants and enthalpy changes for ligand binding to proteins are the consequence of poor experiment design, failure to properly calibrate and test instruments and protocols, lack of controls, errors in solution preparation, and incorrect data analyses. Analysis of a recent report that claimed to have determined the "repeatability, precision, and accuracy of the enthalpies and Gibbs energies of a protein-ligand binding reaction" by ITC is used to illustrate how to improve ITC operations and results. The analysis shows that the reported results are misleading because calorimeters were not calibrated, operating parameters were not optimized, errors were made in solution preparations, and data analysis was not optimized. As a consequence, the results do not provide a valid comparison of the capabilities of the calorimeters included in the study. A proposal that reaction of acetazolamide with carbonic anhydrase II be used as a comparison standard for testing ITCs and procedures is problematic because the binding constant is too large and for several other reasons discussed in the paper. Requirements for obtaining precise and accurate results by ITC are discussed and experimental results are presented to illustrate the precision and accuracy attainable with low volume ITCs. The problem of the blank correction is identified as the limiting factor in obtaining accurate results by ITC.

中文翻译：

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通过ITC获得准确的结果。

通过周到的实验设计和建模以及精心的实验操作，可以通过等温滴定热分析（ITC）获得精确准确的结果。ITC中报告的大量错误导致化学计量的确定，配体与蛋白质结合的平衡常数和焓变的确定是不良的实验设计，无法正确校准和测试仪器和规程，缺乏对照，溶液制备错误以及不正确的结果数据分析。ITC对最近一份声称已确定“蛋白质-配体结合反应的焓和吉布斯能量的重复性，精确度和准确性”的报告进行了分析，以说明如何改善ITC的操作和结果。分析表明，所报告的结果具有误导性，因为未校准量热仪，未优化运行参数，解决方案制备中出现错误以及未优化数据分析。结果，该结果不能提供研究中所包括的量热仪功能的有效比较。提议将乙酰唑酰胺与碳酸酐酶II的反应用作测试ITC和程序的比较标准是有问题的，因为结合常数太大，并且出于本文讨论的其他几个原因。讨论了通过ITC获得精确结果的要求，并给出了实验结果，以说明小体积ITC可获得的精确度和准确性。