Rathore A S, Horváth C
Department of Chemical Engineering, Yale University, New Haven, CT 06520-8286, USA.
Electrophoresis. 1997 Dec;18(15):2935-43. doi: 10.1002/elps.1150181535.
Peptides containing proline residues, except at the N-terminus, exist in cis and trans forms due to the rigidity of the peptidyl-proline bond. Computer-simulated and experimental electropherograms have been used to study the interplay of the electrophoretic migration and the kinetics of the cis-trans interconversion. The measure of the interference by the reaction is the dimensionless Damköhler number (Da) which is the ratio of the migrant's residence time to the characteristic time of reaction in the capillary. The fraction of the trans conformer that is not separable with 90% purity is employed to quantify the extent of overlap between the concentration profiles of the two interconverting migrants and the effect of the various operational variables was examined by simulation. The deterioration of separation, as measured by the degree of overlap, is a quasi sigmoidal function of Da with the selectivity and the intrinsic efficiency of the system as the parameters. At sufficiently low temperatures the interference by reaction kinetics may vanish and the two conformers are separated. At high enough temperatures, when the rate of interconversion is very fast, the two conformers are not separated at all. A simple and rapid method is proposed for estimation of kinetic parameters for the cis-trans isomerization on the basis of data obtained with phenylalanyl-proline dipeptide. The procedure involves determination of the equilibrium constant by measuring equilibrium concentrations of the two conformers using nuclear magnetic resonance (NMR) or capillary zone electrophoresis (CZE) at very low temperature, correlation of the peak shapes in the electropherograms with the Da, and finally, evaluation of the forward rate constants from the assigned Da values in the domain: 0.01 < Da < 0.5. Separations using CZE were performed in the temperature range of 1-40 degrees C by using the Beckman P/ACE unit equipped with an auxiliary cooling system. The kinetic data thus obtained showed good agreement (average error less than 5%) with those measured by NMR.
除了N端外，含有脯氨酸残基的肽由于肽基 - 脯氨酸键的刚性而以顺式和反式形式存在。计算机模拟和实验电泳图已被用于研究电泳迁移与顺 - 反异构化动力学之间的相互作用。反应干扰的量度是无量纲达姆科勒数（Da），它是迁移物在毛细管中的停留时间与反应特征时间的比值。采用纯度无法达到90%分离的反式构象体的比例来量化两种相互转化的迁移物浓度分布之间的重叠程度，并通过模拟研究了各种操作变量的影响。以重叠程度衡量的分离恶化是Da的准S形函数，系统的选择性和固有效率作为参数。在足够低的温度下，反应动力学的干扰可能消失，两种构象体得以分离。在足够高的温度下，当异构化速率非常快时，两种构象体根本无法分离。基于苯丙氨酰 - 脯氨酸二肽获得的数据，提出了一种简单快速的方法来估计顺 - 反异构化的动力学参数。该程序包括在非常低的温度下使用核磁共振（NMR）或毛细管区带电泳（CZE）测量两种构象体的平衡浓度来确定平衡常数，将电泳图中的峰形与Da相关联，最后，从指定的Da值范围：0.01 < Da < 0.5评估正向速率常数。使用配备辅助冷却系统的贝克曼P/ACE装置在1 - 40℃的温度范围内进行CZE分离。由此获得的动力学数据与通过NMR测量的数据显示出良好的一致性（平均误差小于5%）。
