Effects of insulin concentration and self-association on the partitioning of its A-21 cyclic anhydride intermediate to desamido insulin and covalent dimer.
OBJECTIVE
In the pH range 2-5, human insulin degrades via deamidation at the A-21 asn and covalent dimerization. Both products form via a common cyclic anhydride intermediate, a product of intramolecular neucleophilic attack by the A-21 carboxyl terminus. This study examines the influence of [insulin] and self-association on the partitioning of the intermediate to products.


METHODS
Insulin self-association was characterized (pH 2-4) by concentration difference spectroscopy. Deamination rates (pH 2-4) and concurrent rates of covalent dimer formation (pH 4) were determined versus [insulin] at 35 degrees C by initial rates. A mathematical model was developed to account for the overall rate and product composition profile versus pH and [insulin].


RESULTS
Between pH 2-4, insulin self-associates to form non-covalent dimers with a pH independent association constant of 1.8 x 10(4) M-1. The overall rate of degradation is governed by intermediate formation, while product distribution is determined by competition between water and the phe B-1 amino group of insulin for the anhydride. In dilute solutions, deamidation is first-order in [insulin] while covalent dimerization is second-order. Thus, deamidation predominates in dilute solutions but the fraction of covalent dimer formed increases with [insulin]. At high [insulin], self-association inhibits covalent dimer formation, preventing exclusive degradation via this pathway. The model accurately predicts a maximum in covalent dimer formation near pH 4.


CONCLUSIONS
A mechanism is described which accounts for the complex dependence of insulin's degradation rate and product distribution profile on pH (between 2-5) and [insulin]. If these results can be generalized, they suggest that covalent aggregation in proteins may be inhibited by self-association.
D000812:Anhydrides / Q000737:chemistry; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry*; D007700:Kinetics; D008432:Mathematical Computing; D013057:Spectrum Analysis / Q000379:methods
D000812:Anhydrides; D007328:Insulin; C034329:insulin dimers; C018037:insulin, desamido (A21)-