The site-specific oxidation of histidine residues in glycated insulin mediated by copper ions and the relationship between the oxidation sites and the steric conformation of insulin are discussed in this study. Glycated insulin was prepared by incubating native insulin with glucose in 67 mM sodium phosphate, pH 7.5, at 37 degrees C for 30 h. In the presence of micromolar concentrations of Cu2+, glycated insulin was oxidized and its fragmentation or aggregation was detected. Accompanying the fragmentation, new N-termini were generated. The residues in these N-termini were identified as alanine, proline, valine, leucine and isoleucine by comparing dansyl derivatives with standard dansyl-amino acid products. Furthermore, several oxidized products of glycated insulin were isolated using reverse-phase HPLC (P1-P3). From amino acid composition and sequence analyses, it was determined that His10 on the insulin B-chain was modified in each of these peptides, while His5 was also modified in P3. The difference in susceptibility of His10 and His5 to oxidative modification is considered to be due to easier coordination of Cu2+ with His10, which further forms a complex with the Amadori compound at B-chain Phe1 that is vicinal to His10 in the steric conformation of insulin. This complex may generate an active oxygen species, which induces the degradation of the imidazole ring at His10, leading to aggregation or fragmentation of insulin.