Department of Pharmacy, University of Copenhagen, Denmark.
Department of Science and Environment, Roskilde University, Denmark.
Anal Biochem. 2024 Apr;687:115446. doi: 10.1016/j.ab.2023.115446. Epub 2023 Dec 24.
Binding interactions often involve heterogeneous samples displaying a distribution of binding sites that vary in affinity and binding enthalpy. Examples include biological samples like proteins and chemically produced samples like modified cyclodextrins. Experimental studies often ignore sample heterogeneity and treat the system as an interaction of two homogeneous species, i.e. a chemically well-defined ligand binding to one type of site. The present study explores, by simulations and experiments, the impact of heterogeneity in isothermal titration calorimetry (ITC) setups where one of the binding components is heterogeneous. It is found that the standard single-site model, based on the assumption of two homogeneous binding components, provides excellent fits to simulated ITC data when the binding free energy is normally distributed and all sites have similar binding enthalpies. In such cases, heterogeneity can easily go undetected but leads to underestimated binding constants. Heterogeneity in the binding enthalpy is a bigger problem and may result in enthalpograms of increased complexity that are likely to be misinterpreted as two-site binding or other complex binding models. Finally, it is shown that heterogeneity can account for previously observed experimental anomalies. All simulations are accessible in Google Colab for readers to experiment with the simulation parameters.
结合相互作用通常涉及显示在亲和性和结合焓方面有所不同的结合位点分布的多相样品。例如，蛋白质等生物样品和经过化学修饰的环糊精等化学合成样品。实验研究通常忽略样品异质性，并将系统视为两种均相物质的相互作用，即化学定义明确的配体与一种类型的位点结合。本研究通过模拟和实验探讨了在等温滴定量热法（ITC）设置中一个结合成分是多相的情况下异质性的影响。结果发现，当结合自由能呈正态分布且所有位点具有相似的结合焓时，基于两个均相结合成分假设的标准单一位点模型能够很好地拟合模拟的 ITC 数据。在这种情况下，异质性很容易被忽略，但会导致结合常数被低估。结合焓的异质性是一个更大的问题，可能导致焓图变得更加复杂，容易被错误地解释为双位点结合或其他复杂的结合模型。最后，结果表明，异质性可以解释先前观察到的实验异常。所有模拟均可在 Google Colab 中获取，读者可以使用模拟参数进行实验。
