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首页> 外文期刊>Biotechnology Progress >Studies of Lysozyme Binding to Histamine as a Ligand for Hydrophobic Charge Induction Chromatography
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Studies of Lysozyme Binding to Histamine as a Ligand for Hydrophobic Charge Induction Chromatography

机译：溶菌酶与组胺结合作为配体进行疏水电荷诱导色谱的研究

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Histamine was immobilized on Sepharose CL-6B (Sepharose) for use as a ligand of hydrophobic charge induction chromatography (HCIC) of proteins. Lysozyme adsorption onto His-tamine-Sepharose (HAS) 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 HAS 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 (ΔH_(ads), ΔS_(ads), and ΔG_(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 HAS was characterized by a less favorable ΔG_(ads) and an unfavorable ΔS_(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 ΔS_(ads) was offset by a favorable ΔH_(ads), thus exhibiting typical enthalpy-entropy compensation. Moreover, thermodynamic analyses indicated the importance of the dehydration of lysozyme molecule and HAS 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.
机译：组胺被固定在Sepharose CL-6B（Sepharose）上，用作蛋白质的疏水电荷诱导色谱（HCIC）的配体。通过吸附平衡和量热法研究了溶菌酶对组胺-Sepharose（HAS）的吸附，以揭示蛋白质结合的热力学机理。在两个实验中，都检查了盐（硫酸铵和硫酸钠）的影响。吸附等温线表明，HAS在溶菌酶吸附中表现出很高的耐盐性。疏水相互作用和芳族堆积的共同作用很好地说明了该性质。等温线很好地拟合了Langmuir方程，并获得了溶菌酶吸附的平衡参数。另外，通过将溶菌酶溶液滴定到吸附剂悬浮液中的等温滴定量热法获得用于吸附的热力学参数（ΔH_（ads），ΔS_（ads）和ΔG_（ads））。此外，在相同的盐缓冲液中将游离的组胺滴定到溶菌酶溶液中。与溶菌酶与游离组胺的结合相比，溶菌酶吸附到HAS上的特征是ΔG_（ads）较差，而ΔS_（ads）较差，因为组胺通过三碳链间隔基共价附于Sepharose。因此，固定化的组胺只能与蛋白质表面上的残基结合，而不能与疏水口袋中的残基结合，从而导致组胺和溶菌酶之间的取向较差。热力学参数的进一步比较表明，不利的ΔS_（ads）被有利的ΔH_（ads）抵消，因此表现出典型的焓熵补偿。此外，热力学分析表明溶菌酶分子和HAS在吸附过程中脱水的重要性以及在吸附过程中蛋白质的构象变化很大。结果为溶菌酶在新型HCIC材料上的吸附机理提供了清晰的见解。

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