Shank-Retzlaff M L, Sligar S G
Beckman Institute for Advanced Science and Technology, Department of Chemistry, University of Illinois Champaign-Urbana, 61801, USA.
Anal Chem. 2000 Sep 1;72(17):4212-20. doi: 10.1021/ac0001030.
A novel one-step method for determining kinetic rates and equilibrium binding affinities, termed analyte gradient-surface plasmon resonance (AG-SPR) is described. A gradient maker or HPLC pump system is used to produce a gradient so that, under continuous-flow conditions, the concentration of analyte passing over the sensor surface increases linearly with time. The rate at which analyte binds to the immobilized receptors is measured by monitoring the change in the surface plasmon resonance minimum as the analyte concentration increases. Kinetic rates are determined by fitting the data to a modified version of the previously described two-compartment model (Schuck, P.; Minton, A. P. Anal. Biochem. 1996, 240, 262-272). Numerical simulations indicate that AG-SPR results in accurate estimates of both kinetic rates and equilibrium affinities regardless of the intrinsic kinetics of the interaction and can be used for systems under mass transport limitations. Simulations also indicate that AG-SPR can be used to characterize interactions that do not obey pseudo-first-order kinetics due to the presence of a heterogeneous receptor population. Experimentally, the interaction of cytochrome c with cytochrome b5 immobilized on a negatively charged monolayer has been characterized by AG-SPR, and both the specific and the nonspecific interactions were quantitatively analyzed. This new technique is advantageous over traditional SPR methods because it eliminates the need for surface regeneration and is significantly faster than traditional titration experiments.
本文描述了一种用于测定动力学速率和平衡结合亲和力的新型一步法，称为分析物梯度表面等离子体共振（AG-SPR）。使用梯度发生器或高效液相色谱泵系统产生梯度，以便在连续流动条件下，流经传感器表面的分析物浓度随时间线性增加。通过监测随着分析物浓度增加表面等离子体共振最小值的变化，来测量分析物与固定化受体的结合速率。通过将数据拟合到先前描述的双室模型的修改版本（Schuck, P.; Minton, A. P. Anal. Biochem. 1996, 240, 262 - 272）来确定动力学速率。数值模拟表明，无论相互作用的内在动力学如何，AG-SPR都能准确估计动力学速率和平衡亲和力，并且可用于受传质限制的系统。模拟还表明，由于存在异质受体群体，AG-SPR可用于表征不遵循伪一级动力学的相互作用。在实验中，细胞色素c与固定在带负电荷单层上的细胞色素b5的相互作用已通过AG-SPR进行了表征，并对特异性和非特异性相互作用进行了定量分析。这项新技术比传统的SPR方法更具优势，因为它无需表面再生，并且比传统滴定实验快得多。
