Improved insulin stability through amino acid substitution

• Published in: Protein engineering 1992-09, Vol.5 (6), p.519-525
• Main Authors: Brems, David N., Brown, Patricia L., Bryant, Christopher, Chance, Ronald E., Green, L. Kenney, Long, Harlan B., Miller, Alita A., Millican, Rohn, Shields, James E., Frank, Bruce H.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid substitution; amino acids; Biological and medical sciences; Biotechnology; Cystine; Drug Stability; Fundamental and applied biological sciences. Psychology; insulin; Insulin - analogs & derivatives; Insulin - chemistry; insulin stability; Methods. Procedures. Technologies; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Polymers; Protein Conformation; Protein Denaturation; Protein engineering; Protein Structure, Tertiary; stability; substitution; Temperature; Thermodynamics
• ISSN: 1741-0126; 0269-2139; 1741-0134; 1460-213X
• 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 disulfidelinked multimer that was formed through a disulfide interchange reaction which resulted from β-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 inter change 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 correla tion (R2=0.8 and P < 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.