Developing Adsorptive Protein Separations

这是一项关于蛋白质在层析树脂间隙中的吸附和扩散的实验研究。这项研究的主要重点是用核磁共振技术测量树脂中的扩散系数和吸附速率常数。这项技术最早是由PI使用蛋白蛋清溶菌酶开创的。这种酶用氟自旋标记，溶解在没有吸附边界的介质中，并受到射频脉冲的影响。写出了脉冲产生的横向和纵向磁化强度的演化方程。从这些方程中，可以得到作为示踪剂扩散系数的函数的可调焦磁化强度；与磁化强度的实测值进行比较，可以计算扩散系数。该方案为吸附测量提供了一种新的思路。PI认为，当蛋白质在液体中的横向磁化衰减率远小于蛋白质在表面的衰减率，并且吸附的特征时间远长于被吸附物种的横向磁化松弛时间时，控制可重聚焦磁化的方程与非吸附介质中的弛豫相同，吸附使系数重归一化。他们建议首先使用这种技术来获得扩散系数，然后使用模拟回波来测量吸附系数，在模拟回波中，纵向弛豫控制了大部分响应。吸附没有集中在纵向衰减的动力学方程中，因为表面的纵向弛豫衰减的尺度与体相的尺度相同。这项研究将在实验上考察新的吸附材料，特别是小孔尺寸排除树脂中的吸附和扩散，以确定阻碍扩散的影响，然后是离子交换、疏水相互作用和反相系统。将对这些材料进行研究，以期在灌注色谱中实现最终用途。在这项研究的第二部分，蛋白质分子将通过螯合剂被标记上顺磁性离子，以使水中的质子依赖于局部蛋白质浓度。PI打算校准这一效应，并使用它来测量间隙空间中的浓度分布。在研究的第三部分，也是最后一部分，建模工作将针对扩散和吸附的预测，包括孔尺寸分布、吸附动力学和高负载的影响。单独的理论努力将集中在混相驱替前锋的不稳定性上。

This proposal is an experimental study of adsorption and diffusion of proteins within the interstitial spaces of a chromatographic resin. The primary focus of this study is the measurement of the diffusion coefficients and adsorption rate constants in resins by an NMR technique. This technique was first pioneered by the PIs using the protein hen egg white lysozyme. This enzyme was labelled with a fluorine spin label, dissolved in a medium without adsorbing boundaries, and subject to an rf pulse. Equations are written for the evolution of the transverse and longitudinal magnetization imparted by the pulse. From these equations, the magnitudes of the refocussable magnetization can be obtained as a function of the tracer diffusion coefficient; comparisons with the measured values of the magnetization allows for the computation of the diffusion coefficient. The proposal presents a novel idea for the measurement of adsorption. The PIs argue that when the decay rate of the transverse magnetization of the protein in the fluid is much smaller than that for the protein on the surface, and the characteristic time for adsorption is much longer than the time for relaxation of the transverse magnetization of adsorbed species, the equation which governs the refocussable magnetization is identical to that for relaxation in a nonadsorbing medium, with adsorption renormalizing a coefficient. They suggest first using this technique to obtain the diffusion coefficient, and then using a simulated echo, in which longitudinal relaxation governs much of the response, to measure the adsorption coefficient. Adsorption is not lumped in the dynamic equations for the longitudinal decay because the scale for longitudinal relaxation decay on the surface is of the order of that in the bulk. The proposed research will examine experimentally adsorption and diffusion in new adsorbent materials, particularly size excluded resins with small pores in order to determine the effects of hindered diffusion, and then in ion exchange, hydrophobic interaction and reversed phase systems. These materials will be studied with a view towards end-use applications in perfusion chromatography. In a second part of the study, protein molecules will be tagged with paramagnetic ions by means of chelators in order to render the aqueous protons dependent on the local protein concentration. The PIs intend to calibrate this effect, and use it to measure concentration profiles in the interstitial space. In a third and final part of the study, modeling efforts will be directed at prediction of diffusion and adsorption including the effects of pore size distribution, adsorption kinetics, and high loading. Separate theoretical efforts will focus on instabilities at miscible displacement fronts.