Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods
Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods
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DOI：
10.1110/ps.4330102
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发表时间：
2002-05-01
期刊：
影响因子：
8
通讯作者：
Myszka, DG
中科院分区：
文献类型：
--
作者：
Day, YSN;Baird, CL;Myszka, DG
小分子与碳酸酐酶11的结合相互作用被用作模型系统，以比较从基于表面和溶液的生物物理方法确定的反应常数。收集了两种芳基磺酰胺化合物，4-羧基苯磺酰胺（CBS）和5-二甲基氨基-1-萘磺酰胺（DNSA），使用表面等离子体共振，等温滴定量热法和停流荧光结合酶的相互作用数据。我们表明，当表面等离子体共振生物传感器实验小心执行，平衡，热力学和动力学常数从这种基于表面的技术确定匹配那些在溶液中获得。这些结果验证了使用生物传感器技术来收集关于小分子与固定化大分子靶标结合的可靠数据。结合动力学显示，提供更详细的信息复杂的形成比平衡常数。例如，虽然碳酸酐酶11结合DNSA的亲和力比CBS高两倍，但动力学分析显示CBS的解离速率慢四倍。结合态和过渡态热力学的分析也揭示了复合物形成的焓和熵的显着差异。标签要求少，信息含量高。并且表面等离子体共振生物传感器的高通量将使该技术成为表征小分子与酶和受体的相互作用的重要工具。
The binding interactions of small molecules with carbonic anhydrase 11 were used as model systems to compare the reaction constants determined from surface- and solution-based biophysical methods. Interaction data were collected for two arylsulfonamide compounds, 4-carboxybenzenesulfonamide (CBS) and 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), binding to the enzyme using surface plasmon resonance, isothermal titration calorimetry, and stopped-flow fluorescence. We demonstrate that when the surface plasmon resonance biosensor experiments are per-formed with care, the equilibrium, thermodynamic, and kinetic constants determined from this surface-based technique match those acquired in solution. These results validate the use of biosensor technology to collect reliable data on small molecules binding to immobilized macromolecular targets. Binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone. For example, although carbonic anhydrase 11 bound DNSA with twofold higher affinity than CBS, kinetic analysis revealed that CBS had a fourfold slower dissociation rate. Analysis of the binding and transition state thermodynamics also revealed significant differences in the enthalpy and entropy of complex formation. The lack of labeling requirements, high information content. and high throughput of surface plasmon resonance biosensors will make this technology an important tool for characterizing the interactions of small molecules with enzymes and receptors.