Dept. of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P.R. China.
Biotechnol Prog. 2010 Jan-Feb;26(1):134-41. doi: 10.1002/btpr.295.
Histamine was immobilized on Sepharose CL-6B (Sepharose) for use as a ligand of hydrophobic charge induction chromatography (HCIC) of proteins. Lysozyme adsorption onto Histamine-Sepharose (HA-S) was studied by adsorption equilibrium and calorimetry to uncover the thermodynamic mechanism of the protein binding. In both the experiments, the influence of salt (ammonium sulfate and sodium sulfate) was examined. Adsorption isotherms showed that HA-S exhibited a high salt tolerance in lysozyme adsorption. This property was well explained by the combined contributions of hydrophobic interaction and aromatic stacking. The isotherms were well fitted to the Langmuir equation, and the equilibrium parameters for lysozyme adsorption were obtained. In addition, thermodynamic parameters (DeltaH(ads), DeltaS(ads), and DeltaG(ads)) for the adsorption were obtained by isothermal titration calorimetry by titrating lysozyme solutions into the adsorbent suspension. Furthermore, free histamine was titrated into lysozyme solution in the same salt-buffers. Compared with the binding of lysozyme to free histamine, lysozyme adsorption onto HA-S was characterized by a less favorable DeltaG(ads) and an unfavorable DeltaS(ads) because histamine was covalently attached to Sepharose via a three-carbon-chain spacer. Consequently, the immobilized histamine could only associate with the residues on the protein surface rather than those in the hydrophobic pocket, causing a less favorable orientation between histamine and lysozyme. Further comparison of thermodynamic parameters indicated that the unfavorable DeltaS(ads) was offset by a favorable DeltaH(ads), thus exhibiting typical enthalpy-entropy compensation. Moreover, thermodynamic analyses indicated the importance of the dehydration of lysozyme molecule and HA-S during the adsorption and a substantial conformational change of the protein during adsorption. The results have provided clear insights into the adsorption mechanisms of lysozyme onto the new HCIC material.
组胺被固定在 Sepharose CL-6B（Sepharose）上，用作蛋白质疏水电荷诱导层析（HCIC）的配体。通过吸附平衡和量热法研究溶菌酶在组胺-Sepharose（HA-S）上的吸附，以揭示蛋白质结合的热力学机制。在这两个实验中，都考察了盐（硫酸铵和硫酸钠）的影响。吸附等温线表明，HA-S 在溶菌酶吸附中具有较高的耐盐性。这种性质可以通过疏水相互作用和芳环堆积的综合贡献得到很好的解释。等温线很好地符合朗缪尔方程，得到了溶菌酶吸附的平衡参数。此外，通过将溶菌酶溶液滴定到吸附剂悬浮液中，通过等温滴定量热法获得了吸附的热力学参数（ΔH（ads）、ΔS（ads）和ΔG（ads））。此外，在相同盐缓冲液中，将游离组胺滴定到溶菌酶溶液中。与游离溶菌酶与游离组胺的结合相比，溶菌酶吸附到 HA-S 上的特点是ΔG（ads）较小且ΔS（ads）不利，因为组胺通过三碳链间隔物共价连接到 Sepharose 上。因此，固定化组胺只能与蛋白质表面的残基结合，而不能与疏水性口袋中的残基结合，导致组胺与溶菌酶之间的取向不太有利。进一步比较热力学参数表明，不利的ΔS（ads）被有利的ΔH（ads）抵消，从而表现出典型的焓熵补偿。此外，热力学分析表明，在吸附过程中溶菌酶分子和 HA-S 的脱水以及蛋白质的构象发生重大变化非常重要。这些结果为溶菌酶在新型 HCIC 材料上的吸附机制提供了清晰的认识。
