Biophysical Characterization of Macromolecules

动力学速率常数的测定和 生物分子相互作用的平衡常数 生物传感器经常受到缺乏可靠结果的困扰， 特别是对于高亲和力相互作用。我们先前已经 确定了移动的反应物对结合的转运限制 固定在传感器表面的位点作为可能的 生物传感器数据的动力学分析中的错误的根本原因。 我们已经开发出一种实验性的配置， 直接测定平衡结合常数 表面等离子体激元样品处理系统的改进 共振生物传感器我们修改了分析物的供应， 以前通过有限体积的单次注射实现 变成一个有限的微流体回路。在新配置中，我们使用 外部注射泵，用于连续回收样品 在一个封闭的环内，从而提供了一个几乎无限的接触 样品与其固定化位点的时间。这允许 达到相互作用的热力学平衡，甚至 用于非常慢的结合速率和高亲和力相互作用，以及 能够测量平衡结合常数， 平衡滴定法虽然这种技术避免了 为了解释结合动力学并避免相关的伪像， 还消除了在中间的化学再生的需要， 常规的多动力学结合-解吸循环。这 方法在溶菌酶的研究中得到初步应用 与Smith-Gill博士合作的抗体相互作用。中 项目使用传统的光学生物传感器配置， 与Padlan博士和Helm博士的实验室合作， 提出了一种新的动力学速率对比分析方法 常数，并应用于一系列IgE突变体与 用于识别结构上重要的IgE受体 残基使用表面等离子体共振的合作研究 这项技术已经开始与菲什曼博士的实验室， G蛋白相互作用的表征，并与博士。 Sharma关于量化细胞周期蛋白依赖性 激酶。这是校内研究项目的延续 Z01-RR-10485-01 BEI。

The determination of kinetic rate constants and of equilibrium constants of biomolecular interactions using optical biosensors has frequently suffered from lack of reliable results, in particular for high-affinity interactions. We have previously identified transport limitation of the mobile reactant to the binding sites immobilized at the sensor surface as one of the possible underlying causes of error in the kinetic analysis of biosensor data. We have developed an experimental configuration that allows the direct measurement of equilibrium binding constants by a modification of the sample handling system of our surface plasmon resonance biosensor. We have modified the analyte supply, previously achieved through a single injection of limited volume into a finite microfluidics loop. In the new configuration, we use an external syringe pump for the continuous recycling of the sample within a closed loop, thereby providing a virtually unlimited contact time of the sample with its immobilized sites. This allows the attainment of thermodynamic equilibrium of the interaction, even for very slow binding rates and high affinity interactions, and enables the measurement of equilibrium binding constants in an equilibrium titration approach. While this technique avoids the need to interpret the binding kinetics and avoids the related artifacts, it also eliminates the need for chemical regeneration in between the conventional multiple kinetic binding-desorption cycles. This method was used in initial applications in the study of lysozyme antibody interactions in collaboration with Dr. Smith-Gill. In a project using the conventional optical biosensor configuration in collaboration with Dr. Padlan and the laboratory of Dr. Helm, a new method was developed for comparative analysis of kinetic rate constants, and applied to the binding of a series of IgE mutants to the IgE-receptor for the identification of structurally important residues. Collaborative studies using the surface plasmon resonance technique have been started with the laboratories of Dr. Fishman on the characterization of G-protein interactions, and with the Dr. Sharma on the quantification of the interactions of cyclin-dependent kinases. This is a continuation of Intramural Research Project Z01-RR-10485-01 BEI.