Wang Hailin, Wang Zhixin, Lu Meiling, Zou Hanfa
State Key Laboratory of Environmental Chemistry and Eco-toxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.
Anal Chem. 2008 Apr 15;80(8):2993-9. doi: 10.1021/ac702066x. Epub 2008 Mar 21.
Here we present an application of microdialysis sampling for evaluation of the binding kinetics of small molecules to macromolecules. It is label-free, and no immobilization of any interaction partner is required. The method was established by the coupling of a binding reaction with a membrane transport in a miniature and dynamic microdialysis sampling system. A theoretical model was established to describe the quantitative relationship between the binding kinetics of small ligands to macromolecules and the enhanced mass transport of small ligands and was applied to estimate the binding kinetics. To demonstrate the proof-of-principle, we examined the binding kinetics of an abundant plasma protein human serum albumin (HSA) and a representative drug ketoprofen as an example. The primary binding constant of ketoprofen to HSA was estimated as 1.63 (+/-0.12) x 10(6) M(-1). The estimated association and dissociation rate constants (k1 and k(-1)) were about 3.71 x 10(5) M(-1) s(-1) and 0.227 s(-1), respectively. The results suggest a fast binding of ketoprofen to HSA and a fast dissociation of the formed complex, which are consistent with the reversible binding property of drug and HSA (k(-1) in the order of s(-1)). This is the first report on binding-kinetics measurement using microdialysis sampling.
在此，我们展示了微透析采样在评估小分子与大分子结合动力学方面的应用。它无需标记，也无需固定任何相互作用的伙伴。该方法是通过在一个微型动态微透析采样系统中将结合反应与膜转运相结合而建立的。建立了一个理论模型来描述小配体与大分子的结合动力学以及小配体增强的质量传输之间的定量关系，并应用该模型来估计结合动力学。为了证明原理，我们以丰富的血浆蛋白人血清白蛋白（HSA）和代表性药物酮洛芬为例，研究了它们的结合动力学。酮洛芬与HSA的一级结合常数估计为1.63（±0.12）×10⁶ M⁻¹。估计的缔合和解离速率常数（k₁和k⁻¹）分别约为3.71×10⁵ M⁻¹ s⁻¹和0.227 s⁻¹。结果表明酮洛芬与HSA的结合迅速，形成的复合物解离也迅速，这与药物和HSA的可逆结合特性一致（k⁻¹约为s⁻¹量级）。这是关于使用微透析采样进行结合动力学测量的首次报道。
