Ma S, Kálmán F, Kálmán A, Thunecke F, Horváth C
Department of Chemical Engineering, Yale University, New Haven, CT 06520, USA.
J Chromatogr A. 1995 Nov 17;716(1-2):167-82. doi: 10.1016/0021-9673(95)00555-2.
The cis-trans conformers of two dipeptides, Phe-Pro and Leu-Pro, and two opioid heptapeptides containing one or two proline residues were separated by capillary zone electrophoresis (CZE) in borate buffer at low temperatures down to -17 degrees C. At temperatures near ambient, the relaxation time of the cis-trans isomerization is on the time-scale of minutes for the dipeptides and thus commensurate with the migration times in CZE under usual operating conditions. The conformers of both dipeptides could be separated with baseline resolution below 10 degrees C in neat aqueous 100 mM sodium borate (pH 8.4). The conformer peaks on the electropherograms were identified by using authentic samples of the cis and trans forms of Phe-Pro and Leu-Pro that were obtained by reversed-phase HPLC at 0 degree C, validated by NMR spectroscopy and stored in liquid nitrogen. The interplay of the electrophoretic migration and on-column isomerization reaction in CZE of Phe-Pro under various conditions was analyzed in the light of the Damköhler number (Da). The results showed that besides employing low temperature increasing the voltage and/or decreasing the capillary length also reduce the magnitude of Da to bring about the separation of interconverting species. In this work the use of low temperature in this work was preferred due to the experimental simplicity. The separation of cis-trans conformers of two opioid heptapeptides was carried out by CZE at subzero temperatures with aqueous sodium borate containing 23% (v/v) glycerol at pH* 11.3 as measured with a glass electrode. The two conformers of Tyr-Pro-Phe-Asp-Val-Val-Gly-NH2 were baseline separated at -12 degrees C and the four conformers of Tyr-Pro-Phe-Gly-Tyr-Pro-Ser-NH2 due to the presence of two peptidyl-proline bonds in the molecule, were also resolved at -12 degrees C. From the electrophoretic mobilities, the hydrodynamic radii of the cis-trans conformers of the dipeptides Phe-Pro and Leu-Pro were estimated. In both cases, the trans isomers had 1.3 times greater Stokes radii than the cis conformers. This agrees with the observed migration order and molecular modeling results. The hydrodynamic radii of the Phe-Pro conformers were smaller than those of the Leu-Pro isomers despite the lower molecular mass of the latter. The results demonstrate that CZE is suitable for measuring certain molecular properties and suggest that the methods introduced here are applicable to the study of other systems of interconverting conformers.
在低至 -17℃的硼酸盐缓冲液中，通过毛细管区带电泳（CZE）分离了两种二肽（苯丙氨酸 - 脯氨酸和亮氨酸 - 脯氨酸）以及两种含有一个或两个脯氨酸残基的阿片样七肽的顺反异构体。在接近环境温度时，二肽顺反异构化的弛豫时间在分钟时间尺度上，因此与常规操作条件下CZE中的迁移时间相当。在100 mM硼酸钠纯水溶液（pH 8.4）中，低于10℃时两种二肽的异构体均可实现基线分离。通过在0℃下进行反相高效液相色谱法获得苯丙氨酸 - 脯氨酸和亮氨酸 - 脯氨酸顺式和反式形式的真实样品，并通过核磁共振光谱法进行验证，然后储存在液氮中，以此来识别电泳图上的异构体峰。根据达姆科勒数（Da）分析了苯丙氨酸 -脯氨酸在CZE中各种条件下的电泳迁移与柱上异构化反应之间的相互作用。结果表明，除了采用低温外，增加电压和/或减小毛细管长度也会降低Da的大小，从而实现互变物种的分离。在本研究中，由于实验操作简单，因此更倾向于使用低温。在零下温度下，以含有23%（v/v）甘油的硼酸钠水溶液（用玻璃电极测得pH为11.3）为缓冲液，通过CZE分离两种阿片样七肽的顺反异构体。在 -12℃时，酪氨酸 - 脯氨酸 - 苯丙氨酸 - 天冬氨酸 - 缬氨酸 - 缬氨酸 - 甘氨酸 - 氨基的两种异构体实现了基线分离；由于分子中存在两个肽基 - 脯氨酸键，在 -12℃时，酪氨酸 - 脯氨酸 - 苯丙氨酸 - 甘氨酸 - 酪氨酸 - 脯氨酸 - 丝氨酸 - 氨基的四种异构体也得到了分离。根据电泳迁移率，估算了二肽苯丙氨酸 - 脯氨酸和亮氨酸 - 脯氨酸顺反异构体的流体力学半径。在这两种情况下，反式异构体的斯托克斯半径比顺式异构体大1.3倍。这与观察到的迁移顺序和分子模拟结果一致。尽管亮氨酸 - 脯氨酸的分子量较低，但其顺反异构体的流体力学半径却比苯丙氨酸 - 脯氨酸的大。结果表明，CZE适用于测量某些分子性质，并表明本文介绍方法适用于其他互变异构体体系的研究。
