Elucidation of a novel polypeptide cross-link involving 3-hydroxykynurenine

3-Hydroxykynurenine, a metabolite of tryptophan, is a powerful antioxidant and neurotoxin. The neurotoxicity results from the oxidation of 3-hydroxyky nurenine, and hydroxyl radicals, formed via H2O2, may also be implicated [O kuda, S., Nishiyama, N., Saito, H., and Katsuki, H. (1996) Proc. Natl. Acad . Sci. U.S.A. 93, 12553-12558]. Oxidation of o-aminophenols, such as 3-hydr oxykynurenine, also results in the formation of highly reactive quinonimine s. Thus. one possible consequence of 3-hydroxykynurenine oxidation may be c ovalent modification of cellular macromolecules. Such a process could contr ibute to the neurotoxicity and may potentially be important in other tissue s, such as the human lens, where 3-hydroxykynurenine functions as a UV filt er. In this work, we demonstrate that 3-hydroxykynurenine can bind to prote in amino groups and, further, that under oxidative conditions, 3-hydroxykyn urenine can function to cross-link polypeptide chains. The structure of the cross-linked moiety, using the peptide glycyllysine, has been elucidated. The cross-link, which is both colored and fluorescent, involves the peptide alpha-amino groups. Proteins modified by 3-hydroxykynurenine become colore d and fluorescent as well as cross-linked. LC-MS studies indicate that the cross-link is also present in gamma-crystallin, following incubation of thi s lens protein in the presence of 3-hydroxykynurenine. Similar posttranslat ional modifications of lens proteins accompany cataract formation, and know ledge of the precise mode of reaction of 3-hydroxykynurenine with proteins will assist in determining if 3-hydroxykynurenine is involved in degenerati ve conditions in which oxidation of such aminophenols is implicated.