Miller K R, Cistola D P
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.
Mol Cell Biochem. 1993;123(1-2):29-37. doi: 10.1007/BF01076472.
Titration calorimetry has been evaluated as a method for obtaining binding constants and thermodynamic parameters for the cytosolic fatty acid- and lipid-binding proteins. An important feature of this method was its ability to accurately determine binding constants in a non-perturbing manner. The equilibrium was not perturbed, since there was no requirement to separate bound and free ligand in order to obtain binding parameters. Also, the structure of the lipid-protein complex was not perturbed, since native ligands were used rather than non-native analogues. As illustrated for liver fatty acid-binding protein, the method distinguished affinity classes whose dissociation constants differed by an order of magnitude or less. It also distinguished endothermic from exothermic binding reactions, as illustrated for the binding of two closely related bile salts to ileal lipid-binding protein. The main limitations of the method were its relatively low sensitivity and the difficulty working with highly insoluble ligands, such as cholesterol or saturated long-chain fatty acids. However, the signal-to-noise ratio was improved by manipulating the buffer conditions, as illustrated for oleate binding to rat intestinal fatty acid binding protein. Binding parameters are reported for oleate interactions with several wild-type and mutant lipid-binding proteins from intestine. Where possible, the binding parameters obtained from calorimetry were compared with results obtained from fluorescence and Lipidex binding assays of comparable systems.
滴定热分析法已被评估为一种获取胞质脂肪酸结合蛋白和脂质结合蛋白结合常数及热力学参数的方法。该方法的一个重要特点是能够以非干扰方式准确测定结合常数。由于无需分离结合态和游离态配体即可获得结合参数，所以平衡未受干扰。此外，由于使用的是天然配体而非非天然类似物，脂质 - 蛋白质复合物的结构也未受干扰。如肝脂肪酸结合蛋白所示，该方法能区分解离常数相差一个数量级或更小的亲和力类别。它还能区分吸热和放热结合反应，如两种密切相关的胆汁盐与回肠脂质结合蛋白的结合所示。该方法的主要局限性在于其相对较低的灵敏度以及处理高度不溶性配体（如胆固醇或饱和长链脂肪酸）的困难。然而，通过控制缓冲条件可提高信噪比，如油酸与大鼠肠道脂肪酸结合蛋白的结合所示。报告了油酸与几种来自肠道的野生型和突变型脂质结合蛋白相互作用的结合参数。在可能的情况下，将量热法获得的结合参数与类似系统的荧光和Lipidex结合测定结果进行了比较。
