Laboratory of Mechanistic Glycobiology Department of Chemistry, Michigan Technological University, Houghton, MI, USA.
Health Research Institute, Michigan Technological University, Houghton, MI, USA.
Methods Mol Biol. 2022;2442:169-185. doi: 10.1007/978-1-0716-2055-7_10.
Isothermal titration microcalorimetry (ITC) can directly determine the thermodynamic binding parameters of biological molecules including affinity constant, binding stoichiometry, heat of binding (enthalpy) and indirectly the entropy, and free energy of binding. ITC has been extensively used to study the binding of lectins to mono- and oligosaccharides, but limitedly in applications to lectin-glycoprotein interactions. Inherent experimental challenges to ITC include sample precipitation during the experiment and relative high amount of sample required, but careful design of experiments can minimize these problems and allow valuable information to be obtained. For example, the thermodynamics of binding of lectins to multivalent globular and linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules leading to increased affinity. Importantly, the mechanism of binding of lectins to mucins appears similar to that for a variety of protein ligands binding to DNA. Recent results also show that high-affinity lectin-mucin cross-linking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects that facilitate binding interactions.
等温热滴定微量量热法（ITC）可以直接测定生物分子的热力学结合参数，包括亲和力常数、结合化学计量、结合热（焓），并间接地测定熵和结合自由能。ITC 已广泛用于研究凝集素与单糖和寡糖的结合，但在凝集素-糖蛋白相互作用中的应用有限。ITC 实验中存在一些固有的实验挑战，包括实验过程中的样品沉淀和相对较高的样品需求，但通过仔细设计实验可以最小化这些问题，并获取有价值的信息。例如，已经描述了凝集素与多价球形和线性糖蛋白（粘蛋白）结合的热力学。结果与动态结合机制一致，在该机制中，凝集素结合并在这些分子中的碳水化合物表位之间跳跃，从而增加亲和力。重要的是，凝集素与粘蛋白的结合机制似乎与各种蛋白质配体与 DNA 的结合机制相似。最近的结果还表明，高亲和力的凝集素-粘蛋白交联相互作用是由结合和跳跃机制相关的有利结合熵驱动的。这些结果表明，配体与生物聚合物的结合通常可能涉及一种共同的机制，涉及增强的熵效应，从而促进结合相互作用。
