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. Study holds ProTherm entries: 9845, 9846, 9847, 9848, 9849, 9850, 9851, 9852, 9853, 9854, 9855, 9856, 9857, 9858, 9859, 9860, 9861, 9862, 9863 Extra Details: additive : DTT(25 mM), amino acid substiution; insulin stability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:38 p.m.
|Number of data points||38|
|Proteins||Insulin ; Insulin|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence A:GIVEQCCTSICSLYQLENYCN/B:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/C:GIVEQCCTSICSLYQLENYCN/D:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/E:GIVEQCCTSICSLYQLENYCN/F:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/G:GIVEQCCTSICSLYQLENYCN/H:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/I:GIVEQCCTSICSLYQLENYCN/J:FVNQHLCGSHLVEALYLVCGERGFFYTPKT/K:GIVEQCCTSICSLYQLENYCN/L:FVNQHLCGSHLVEALYLVCGERGFFYTPKT|