Role of self-association and intramolecular catalysis in the chemical stability of human insulin at low pH

Update Item Information
Title Role of self-association and intramolecular catalysis in the chemical stability of human insulin at low pH
Publication Type dissertation
School or College College of Pharmacy
Department Pharmacotherapy
Author Darrington, Richard Todd
Date 1993-12
Description Advances in biotechnology have resulted in a dramatic increase in the number of proteins currently under development as therapeutic agents. Developing suitable formulations for these agents has been hampered due to the propensity of proteins to degrade via acyl transfer reactions (primarily deamidation), and covalent aggregate formation, processes which are frequently poorly understood in proteins. However, in order to acquire a quantitative, mechanistic understanding of these degradation pathways the self-association behavior of the protein must also be considered. Using the method of initial rates and anhydride trapping the mechanisms of insulin degradation were explored at 35°C and pH 2-5. Insulin was shown to either deamidate at the A-chain C-terminal asparagine or form a covalent dimer linkage between the A-chain C-terminal asparagine and the N-terminal amino group of the B-chain phenylalanine. Anhydride trapping studies indicated that both deamidation and dimerization involve the rate limiting formation of an anhydride intermediate, the result of intramolecular nucleophilic catalysis by the un-ionized C-terminus of the A-chain. At pH 2.0 the anhydride partitioned exclusively to deamidated insulin. With increasing pH deamidation decreased while dimerization increased, suggesting that the B-chain phenylalanine successfully competes with water for the intermediate. However, the pH-rate behavior of dimerization could not be adequately described when only the fraction of free amine was considered, suggesting that dimerization may be influenced by additional ionizable groups. Taking into account charge states near the reaction site resulted in a model which was superior to others investigated in describing the observed behavior. The role of self-association in the formation and partitioning of the anhydride was investigated utilizing concentration difference spectroscopy and kinetic methods. Results indicated that under the conditions of this study insulin exists as an equilibrium mixture of monomer and dimer and that the association constant was equivalent from pH 2-4. Self-association slightly increased the intermediate formation at pH 2.0-3.0 but had no effect at pH 4.0. However, self-association significantly influenced partitioning of the anhydride, inhibiting dimerization at pH 4.0 suggesting that the ability of the B-chain phenylalanine to successfully compete with water is reduced upon self-association. Modeling of the observed behavior indicated that the reactivity of monomer-associated interactions was independent of whether or not it is the nucleophile or the anhydride that is associated, suggesting that insulin self-association influences dimerization via nonspecific rather than conformational effects.
Type Text
Publisher University of Utah
Subject Therapeutic Use
Subject MESH Insulin; Proteins; Diamines
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "The role of self-association and intramolecular catalysis in the chemical stability of human insulin at low pH". Spencer S. Eccles Health Sciences Library. Print version of "The role of self-association and intramolecular catalysis in the chemical stability of human insulin at low pH". available at J. Willard Marriott Library Special Collection. QP6.5 1993 .D37.
Rights Management © Richard Todd Darrington.
Format application/pdf
Format Medium application/pdf
Format Extent 2,828,010 bytes
Identifier undthes,4494
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
Master File Extent 2,828,038 bytes
ARK ark:/87278/s6f47qxc
Setname ir_etd
ID 191037
Reference URL https://collections.lib.utah.edu/ark:/87278/s6f47qxc
Back to Search Results