Using Capillary Electrophoresis To Follow the Acetylation of the Amino Groups of Insulin and To Estimate Their Basicities

Jinming Gao, Milan Mrksich, Frank A. Gomez, George M. Whitesides

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Abstract

Capillary electrophoresis (CE) is an analytical method that is useful for investigating processes that modify the charge of proteins. This paper explores the ability of CE to rationalize charges and electrophoretic mobilities of a simple protein—insulin and its acylated derivatives—as a function of pH. Insulin is a peptide hormone (MW = 5700) that has two-amino groups (Gα and ) and one ε-amino group (Kε). Treatment of insulin with acetic anhydride affords seven derivatives that differ in the sites of acetylation of the three amino groups. Analysis of the pH dependence of the electrophoretic mobilities of these derivatives gives pKa values for the two N-terminal ammonium groups: pKa (Gα) = 8.4; pKa (Fα) = 7.1. Values of the total charge of insulin estimated from electrophoretic mobility differ from those estimated from values of pKa for its ionizable groups by less than 0.5 unit for both bovine and human insulins over the range of pH from 5.5 to 9.5. Analysis of the concentration dependence of the electrophoretic mobility of insulin yields a lower limit for the association constant for dimerization of insulin of KD ≥ 6 × 103 M-1 (25 mM tris and 192 mM Gly, pH 8.4). Studies of electrophoretic mobility as a function of pH and extent of acetylation of amino groups rationalize the charge of insulin in detail. The sensitivity of CE to charge permits the quantitative study of electrostatic properties of proteins in solution. Insulin is a useful small-protein model with which to investigate phenomena in electrophoresis.

Original languageEnglish (US)
Pages (from-to)3093-3100
Number of pages8
JournalAnalytical chemistry
Volume67
Issue number18
DOIs
StatePublished - Jan 1 1995

ASJC Scopus subject areas

  • Analytical Chemistry

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