Comparisons and Applications of Functional Equations for the Calculation of the Protein-Ligand Binding Constant Based on Fluorescence Spectral Data
ZHANG Jing1, GAO Xuan1, 2, JIN Liang1, WANG Hong-hui1, ZHOU Xi-ping1
1. Key Laboratory of Estuarine Ecological Security and Environmental Health, School of Environmental Science and Engineering, Xiamen University Tan Kah Kee College, Zhangzhou 363105, China
2. College of Environment and Ecology, Xiamen University, Xiamen 361102, China
Abstract：The binding interactions of proteins with ligands play important roles in the physiological and biochemical processes of organisms. Thus it is a research hotspot in many fields. A key objective of the investigations on Protein-Ligand binding is to obtain their binding constant (Kb) for evaluating the strength of their binding. Fluorescence spectroscopy is widely used in Protein-Ligand binding studies as its high sensitivity, convenience, rapidity and low cost. When applying fluorescence spectroscopy, it is a key step to obtain the Kb value through mathematical analysis of fluorescence data with the aid of a functional equation. However, due to the different application range of different equations, the Kb values obtained by different equations for the same system are often varied. This work discussed the derivation process and the application conditions of the functional equations for calculating the Kb value of Protein-Ligand binding using fluorescence spectral data. Based on this, we summarized the optimal equation for the calculation of the Kb value for Protein-Ligand binding modes of 1∶1 and 1∶n (n≥2) under different preconditions. The discussion suggested that the selection of the optimal equation is based on two preconditions: a: Whether the Protein-Ligand complex is fluorescent; b: Whether the ligand concentration is much higher than the protein concentration. On the other hand, the human serum albumin (HSA)-Norfloxacin (NFX) binding system was used as a model, and the difference of the Kb values obtained by the fitting of different functional equations was compared and the corresponding reasons were analyzed. The results showed that HSA bound with NFX in 1∶1 mode, and the Kb value was calculated to be 5.0×104 L·mol-1 at 298 K using the most appropriate equation (S12) for this system. However, the Kb values obtained by equation (S6) and (S24) were 28.8% and 48.6% higher than that by equation (S12) respectively, and the value obtained by equation (S17) was about two orders of magnitude higher. This intuitively showed the effect of choosing the inapplicable equation on the final result. In addition, the Kb value obtained by fluorescence spectroscopy in the present work was compared with those obtained by other techniques, indicating the relative reliability of fluorescence spectroscopy for the calculation of the Kb value in Protein-Ligand binding, and also pointing out the limitations of this method. This work warns us that when obtaining the Kb value of Protein-Ligand binding using fluorescence spectral data, it is very necessary to select the most suitable functional equation according to the preconditions (i.e. whether the hypothesis a and/or b are true or not), which will determine the reliability of the obtained results.
张 静，高 煊，金 亮，王鸿辉，周细平. 基于荧光数据计算蛋白质-配体结合常数的方程的对比及应用研究[J]. 光谱学与光谱分析, 2020, 40(11): 3494-3498.
ZHANG Jing, GAO Xuan, JIN Liang, WANG Hong-hui, ZHOU Xi-ping. Comparisons and Applications of Functional Equations for the Calculation of the Protein-Ligand Binding Constant Based on Fluorescence Spectral Data. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2020, 40(11): 3494-3498.
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