Determination of the cis-trans Isomerization Barriers of L-Alanyl-L-Proline in Aqueous Solutions and at Water/Hydrophobic Interfaces by On-Line Temperature-Jump Relaxation HPLC and Dynamic On-Column Reaction HPLC
Determination of the cis-trans Isomerization Barriers of L-Alanyl-L-Proline in Aqueous Solutions and at Water/Hydrophobic Interfaces by On-Line Temperature-Jump Relaxation HPLC and Dynamic On-Column Reaction HPLC
复制标题
通过在线温跃弛豫 HPLC 和动态柱上反应 HPLC 测定水溶液中和水/疏水界面处 L-丙氨酰-L-脯氨酸的顺反异构化势垒
DOI：
10.1021/acs.analchem.5b02488
复制
发表时间：
2015
影响因子：
7.4
通讯作者：
Shingo Saito
中科院分区：
文献类型：
--
作者：
Masami Shibukawa;Ayaka Miyake;Sayaka Eda;Shingo Saito
脯氨酸反式异构化在蛋白质折叠的速率决定步骤中起着关键作用。因此，它是非常重要的，以了解环境的影响，不仅是本体溶液，但也微环境，如界面，对脯氨酸肽的异构化反应。在这里，我们提出了两种HPLC方法的动力学和平衡参数的测量异构化反应在本体溶液和液/固界面。在线温度跳跃弛豫HPLC（T-jump HPLC）允许通过监测反应两侧纯异构体转化的整个时间过程来测定本体溶液中异构化的正向和反向速率常数，与其他HPLC和毛细管区带电泳以及光谱和量热方法相反，这些方法使用异构体的混合物。然后，我们可以根据动态柱上反应HPLC和T-jump HPLC获得的动力学数据确定肽在液/固界面的顺转异构化障碍。我们观察到，在烷基键合的二氧化硅和聚（苯乙烯-二乙烯基苯）共聚物树脂的表面，在水中的肽键forl-丙氨酰-l-脯氨酸（Ala-Pro）周围的相互转化被加速，这是由显着降低活化焓引起的。量子力学计算结果表明，Ala-Pro在水/疏水界面上的平衡向异构化方向移动以及Ala-Pro的快速异构化是由于疏水材料表面的界面水的极性低于本体水的极性所致。
Prolinecis–transisomerization is known to play a key role in the rate-determining steps of protein folding. It is thus very important to understand the influence of environments, not only bulk solutions but also microenvironments such as interfaces, on the isomerization reaction of proline peptides. Here we present two HPLC methods for measurements of kinetic and equilibrium parameters for the isomerization reactions in bulk solutions and at liquid/solid interfaces. On-line temperature-jump relaxation HPLC (T-jump HPLC) allows the determination of forward and reverse rate constants of the isomerization in a bulk solution by monitoring the whole time course of conversion of pure isomers from both sides of the reaction, in contrast to other HPLC and capillary zone electrophoresis as well as spectrometric and calorimetric methods, which use a mixture of the isomers. We can then determinecis–transisomerization barriers of the peptide at liquid/solid interfaces from the kinetic data obtained by dynamic on-column reaction HPLC and T-jump HPLC. We observed that the interconversion around the peptide bond forl-alanyl-l-proline (Ala-Pro) in water is accelerated at the surfaces of an alkyl-bonded silica and a poly(styrene–divinylbenzene) copolymer resin, and this is caused by a remarkable decrease in the enthalpy of activation. The molecular structures of thecisandtransforms of Ala-Pro estimated by quantum mechanics calculation reveal that an equilibrium shift toward thecisform as well as the rapid isomerization of Ala-Pro at the water/hydrophobic interfaces can be attributed to the lower polarity of the interfacial water at the surfaces of the hydrophobic materials compared to that of bulk water.