Sitkowski Jerzy, Bocian Wojciech, Bednarek Elżbieta, Urbańczyk Mateusz, Koźmiński Wiktor, Borowicz Piotr, Płucienniczak Grażyna, Łukasiewicz Natalia, Sokołowska Iwona, Kozerski Lech
National Medicines Institute, Chełmska 30, 00-725, Warsaw, Poland.
Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, 02-089, Warsaw, Poland.
J Biomol NMR. 2018 Jun;71(2):101-114. doi: 10.1007/s10858-018-0197-y. Epub 2018 Jun 12.
The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled C,N human insulin in water, both in neat, with zinc ions only, and in pharmaceutical formulation, containing only m-cresol as phenolic ligand, glycerol and zinc ions. The results show the dominant role of the pH parameter and the concentration on aggregation. The diffusion coefficient Dav was used for monitoring the overall average state of oligomeric ensemble in solution. The analysis of the experimental data of diffusion measurements, using the direct exponential curve resolution algorithm (DECRA) allows suggesting the two main components of the oligomeric ensemble. The 3D HSQC-iDOSY, (diffusion ordered HSQC) experiments performed on C, N-fully labeled insulin at the two pH values, 4 and 7.5, allow for the first time a more detailed experimental observation of individual components in the ensemble. The discussion involves earlier static and dynamic laser light scattering experiments and recent NMR derived translational diffusion results. The results bring new informations concerning the preparation of pharmaceutical formulation and in particular a role of Zn ions. They also will enable better understanding and unifying the results of studies on insulin misfolding effects performed in solution by diverse physicochemical methods at different pH and concentration.
在水中对未标记的以及均匀标记了碳、氮的人胰岛素进行了核磁共振衍生的平移扩散系数测定，实验条件包括纯胰岛素、仅添加锌离子的情况，以及含有间甲酚作为酚类配体、甘油和锌离子的药物制剂。结果表明pH参数和浓度对聚集起主要作用。扩散系数Dav用于监测溶液中寡聚体整体的平均状态。利用直接指数曲线分辨算法（DECRA）对扩散测量的实验数据进行分析，可推断出寡聚体整体的两个主要成分。在4和7.5这两个pH值下，对完全标记了碳、氮的胰岛素进行的3D HSQC-iDOSY（扩散排序HSQC）实验，首次使得对整体中各个成分进行更详细的实验观察成为可能。讨论涉及早期的静态和动态激光光散射实验以及近期核磁共振衍生的平移扩散结果。这些结果为药物制剂的制备带来了新信息，尤其是锌离子的作用。它们还将有助于更好地理解并统一在不同pH值和浓度下通过多种物理化学方法在溶液中进行的胰岛素错误折叠效应研究的结果。
