title: Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides.
abstract: Superoxide reacts rapidly with other radicals, but these reactions have received little attention in the context of oxidative stress. For tyrosyl radicals, reaction with superoxide is 3-fold faster than dimerization, and forms the addition product tyrosine hydroperoxide. We have explored structural requirements for hydroperoxide formation using tyrosine analogues and di- and tri-peptides. Superoxide and phenoxyl radicals were generated using xanthine oxidase, peroxidase and the respective tyrosine derivative, or by gamma-radiation. Peroxides were measured using FeSO4/Xylenol Orange. Tyrosine and tyramine formed stable hydroperoxides, but N-acetyltyrosine and p-hydroxyphenylacetic acid did not, demonstrating a requirement for a free amino group. Using [14C]tyrosine, the hydroperoxide and dityrosine were formed at a molar ratio of 1.8:1. Studies with pre-formed hydroperoxides, and measurements of substrate losses, indicated that, in the absence of a free amino group, reaction with superoxide resulted primarily in restitution of the parent compound. With dipeptides, hydroperoxides were formed only on N-terminal tyrosines. However, adjacent lysines promoted hydroperoxide formation, as did addition of free lysine or ethanolamine. Results are compatible with a mechanism [d'Alessandro, Bianchi, Fang, Jin, Schuchmann and von Sonntag (2000) J. Chem. Soc. Perkin Trans. II, 1862-1867] in which the phenoxyl radicals react initially with superoxide by addition, and the intermediate formed either releases oxygen to regenerate the parent compound or is converted into a hydroperoxide. Amino groups favour hydroperoxide formation through Michael addition to the tyrosyl ring. These studies indicate that tyrosyl hydroperoxides should be formed in proteins where there is a basic molecular environment. The contribution of these radical reactions to oxidative stress warrants further investigation.
authors.affiliation: Department of Pathology, Christchurch School of Medicine and Health Sciences, P.O. Box 4345, Christchurch, New Zealand. christine.winterbourn@chmeds.ac.nz
authors.forename: Christine C
authors.initials: CC
authors.lastname: Winterbourn
authors.forename: Helena N
authors.initials: HN
authors.lastname: Parsons-Mair
authors.forename: Silvia
authors.initials: S
authors.lastname: Gebicki
authors.forename: Janusz M
authors.initials: JM
authors.lastname: Gebicki
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Davies
mesh_terms: D000588:Amines / Q000737:chemistry; D019281:Dimerization; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000737:chemistry*; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry*; D013481:Superoxides / Q000737:chemistry*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: D000588:Amines; D005609:Free Radicals; D010455:Peptides; D013481:Superoxides; D014443:Tyrosine; D006861:Hydrogen Peroxide; C007543:dityrosine