The role of intramolecular nucleophilic catalysis and the effects of self-association on the deamidation of human insulin at low pH.

The influence of intramolecular catalysis and self-association on the kinetics of deamidation at the A-21 Asn residue of human insulin was explored at low pH and 35 degrees C. Observed rate constants of overall insulin degradation were determined as a function of pH over a pH range of 2.0-5.0 and as a function of total insulin concentration between pH 2.0-4.0. The pH-rate behavior of both monomeric and associated insulin degradation from pH 2.0 to 5.0 indicated intramolecular catalysis by the unionized carboxyl terminus of the A chain. Anhydride trapping with aniline at pH 3.0 provided evidence supporting the formation of a cyclic anhydride intermediate in the rate limiting step indicative of intramolecular nucleophilic catalysis. Insulin in the presence of aniline at low pH formed two anilide products, A-21 N delta 2-phenyl asparagine and N delta 2-phenyl aspartic acid human insulin, at the expense of desamido A-21 formation, consistent with the partitioning of a common intermediate. Self-associated insulin degraded at a rate approximately 2.5 times greater than that of the monomer at pH 2.0 and pH 3. However, self-association had a negligible or slight stabilizing effect on insulin decomposition at pH 4.0. An apparent downward shift in the pKa of the carboxyl terminus of approximately 0.75 units upon self-association and a catalytic rate constant which increases with -COOH acidity are proposed to account for these observations.