Brems D N, Brown P L, Bryant C, Chance R E, Green L K, Long H B, Miller A A, Millican R, Shields J E, Frank B H
Parenteral Products Research and Development, Eli Lilly & Co., Indianapolis, IN 46285.
Protein Eng. 1992 Sep;5(6):519-25. doi: 10.1093/protein/5.6.519.
Insulin analogs designed to decrease self-association and increase absorption rates from subcutaneous tissue were found to have altered stability. Replacement of HB10 with aspartic acid increased stability while substitutions at B28 and/or B29 were either comparable to insulin or had decreased stability. The principal chemical degradation product of accelerated storage conditions was a disulfide-linked multimer that was formed through a disulfide interchange reaction which resulted from beta-elimination of the disulfides. The maintenance of the native state of insulin was shown to be important in protecting the disulfides from reduction by dithiothreitol and implicitly from the disulfide interchange reaction that occurs during storage. To understand how these amino acid changes alter chemical stability, the intramolecular conformational equilibria of each analog was assessed by equilibrium denaturation. The Gibbs free energy of unfolding was compared with the chemical stability during storage for over 20 analogs. A significant positive correlation (R2 = 0.8 and P less than 0.0005) exists between the conformational stability and chemical stability of these analogs, indicating that the chemical stability of insulin's disulfides is under the thermodynamic control of the conformational equilibria.
旨在减少自身缔合并提高皮下组织吸收速率的胰岛素类似物被发现具有改变的稳定性。用天冬氨酸取代HB10可提高稳定性，而在B28和/或B29处的取代与胰岛素相当或稳定性降低。加速储存条件下的主要化学降解产物是一种通过二硫键交换反应形成的二硫键连接的多聚体，该反应由二硫键的β消除引起。胰岛素天然状态的维持对于保护二硫键不被二硫苏糖醇还原以及隐含地防止储存期间发生的二硫键交换反应很重要。为了理解这些氨基酸变化如何改变化学稳定性，通过平衡变性评估了每个类似物的分子内构象平衡。将20多种类似物展开的吉布斯自由能与储存期间的化学稳定性进行了比较。这些类似物的构象稳定性和化学稳定性之间存在显著的正相关（R2 = 0.8，P小于0.0005），表明胰岛素二硫键的化学稳定性受构象平衡的热力学控制。
