Developing Adsorptive Protein Separations

该方案是一项实验研究， 蛋白质在细胞间隙内的扩散 层析树脂 本研究的主要重点是 扩散系数和吸附速率的测定 通过NMR技术测定树脂中的常数。 该技术 首先由PI使用蛋白质鸡蛋白色 溶菌酶 这种酶用氟自旋标记 标记，溶解在介质中，没有吸附边界，和 受到RF脉冲。 方程是为 横向和纵向磁化的演变 由脉冲传递。 根据这些方程， 可重聚焦磁化强度的可重聚焦磁化强度可以作为 示踪剂扩散系数的函数;与 磁化强度的测量值允许 扩散系数的计算。 该提案 提出了一个新的想法，吸附测量。 的 PI认为，当横截面的衰减率 流体中蛋白质的磁化强度远小于 对于表面的蛋白质， 吸附的时间比吸附的时间长得多。 吸附态横向磁化弛豫 物种，控制可重新聚焦的方程 磁化强度与弛豫相同， 非吸附介质，吸附重整化a 系数 他们建议首先使用这种技术， 获得扩散系数，然后使用模拟的 回声，其中纵向弛豫支配大部分的 响应，以测量吸附系数。 吸附 在纵向动力学方程中没有集中 因为纵向弛豫的尺度在 表面与本体中的表面具有相同的量级。 拟议 研究将通过实验研究吸附和扩散 在新的吸附材料，特别是上胶排除树脂中， 为了确定阻碍的影响， 扩散，然后在离子交换，疏水相互作用 和反相系统。 这些材料将被研究 着眼于灌注中的最终应用 层析 在研究的第二部分，蛋白质分子将被 通过螯合剂标记顺磁离子， 使水质子依赖于局部蛋白质 浓度. PI打算校准这种影响， 用它来测量间隙中的浓度分布 空间 在研究的第三部分也是最后一部分， 将努力预测扩散， 吸附包括孔径分布的影响， 吸附动力学和高负载。 分离理论 工作重点将放在混相驱替的不稳定性上 战线

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.