Polypeptide modification and cross-linking by oxidized 3-hydroxykynurenine

3-Hydroxykynurenine (3OHKyn) is present in the mammalian lens as a UV filte r and is formed from kynurenine in the tryptophan metabolic pathway. 3OHKyn is a readily autoxidized o-aminophenol which binds to proteins in vitro. T he lens, particularly its central region, the nucleus, becomes increasingly oxidized with age. Under such conditions, the oxidation products of 3OHKyn may bind to lens proteins and contribute to nuclear cataract formation. Th e purpose of this study was to determine the structures of in vitro reactio n products of 3OHKyn with model peptides as a general model for 3OHKyn modi fication of proteins. 3OHKyn was incubated with the dipeptide glycylglycine (GG) and the tetrapeptide tuftsin (sequence TKPR) under oxidizing conditio ns, and the reaction products were characterized by a variety of spectrosco pic techniques. The major 3OHKyn-GG reaction product involves formation of a benzimidazole moiety between the GG N-terminus and the oxidized amino and /or phenol groups of 3OHKyn. In contrast, tuftsin, which has an N-terminal threonine, forms predominantly a cross-linked dimer with oxidized 3OHKyn. T his product is analogous in structure to the dimeric reaction product, quin ilinobenzoxamine, formed between oxidized 3OHKyn and glycyllysine [Aquilina , J. A., et al. (1999) Biochemistry 38, 11455-11464], which contains a benz oxazole moiety. The identification of a tuftsin dimer suggests that 3OHKyn can react with any peptide having a free cr-amino group, via a general side chain elimination mechanism. The identification of both benzimidazole and benzoxazole adducts in peptides with a free N-terminus suggests that peptid e amino groups can react initially at either the aromatic amino or hydroxyl group of oxidized 3OHKyn. The proportion of each adduct may change, howeve r, depending on the amino acid sequence at the N-terminus.