Chemical modifications in aggregates of recombinant human insulin induced by metal-catalyzed oxidation: covalent cross-linking via michael addition to tyrosine oxidation products.

ABSTRACT:

Purpose

To elucidate the chemical modifications in covalent aggregates of recombinant human insulin induced by metal catalyzed oxidation (MCO).

Methods

Insulin was exposed for 3 h at room temperature to the oxidative system copper(II)/ascorbate. Chemical derivatization with 4-(aminomethyl) benzenesulfonic acid (ABS) was performed to detect 3,4-dihydroxyphenylalanine (DOPA) formation. Electrospray ionization-mass spectrometry (ESI-MS) was employed to localize the amino acids targeted by oxidation and the cross-links involved in insulin aggregation. Oxidation at different pH and temperature was monitored with size exclusion chromatography (SEC) and ESI-MS analysis to further investigate the chemical mechanism(s), to estimate the aggregates content and to quantify DOPA in aggre

SUBMITTER: Torosantucci R 

PROVIDER: S-EPMC3399080 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature