title: Production of lysozyme and lysozyme-superoxide dismutase dimers bound by a ditryptophan cross-link in carbonate radical-treated lysozyme.
abstract: Despite extensive investigation of the irreversible oxidations undergone by proteins in vitro and in vivo, the products formed from the oxidation of Trp residues remain incompletely understood. Recently, we characterized a ditryptophan cross-link produced by the recombination of hSOD1-tryptophanyl radicals generated from attack of the carbonate radical produced during the bicarbonate-dependent peroxidase activity of the enzyme. Here, we examine whether the ditryptophan cross-link is produced by the attack of the carbonate radical on proteins other than hSOD1. To this end, we treated hen egg white lysozyme with photolytically and enzymatically generated carbonate radical. The radical yields were estimated and the lysozyme modifications were analyzed by SDS-PAGE, western blot, enzymatic activity and MS/MS analysis. Lysozyme oxidation by both systems resulted in its inactivation and dimerization. Lysozyme treated with the photolytic system presented monomers oxidized to hydroxy-tryptophan at Trp(28) and Trp(123) and N-formylkynurenine at Trp(28), Trp(62) and Trp(123). Lysozyme treated with the enzymatic system rendered monomers oxidized to N-formylkynurenine at Trp(28). The dimers were characterized as lysozyme-Trp(28)-Trp(28)-lysozyme and lysozyme-Trp(28)-Trp(32)-hSOD1. The results further demonstrate that the carbonate radical is prone to causing biomolecule cross-linking and hence, may be a relevant player in pathological mechanisms. The possibility of exploring the formation of ditryptophan cross-links as a carbonate radical biomarker is discussed.
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo.
authors.forename: Verônica
authors.initials: V
authors.lastname: Paviani
authors.affiliation: Departamento de Química e Exatas, Universidade Estadual do Sudoeste da Bahia.
authors.forename: Raphael F
authors.initials: RF
authors.lastname: Queiroz
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo.
authors.forename: Emerson F
authors.initials: EF
authors.lastname: Marques
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo.
authors.forename: Paolo
authors.initials: P
authors.lastname: Di Mascio
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo. Electronic address: oaugusto@iq.usp.br.
authors.forename: Ohara
authors.initials: O
authors.lastname: Augusto
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002254:Carbonates / Q000737:chemistry* / Q000378:metabolism; D002645:Chickens; D003432:Cross-Linking Reagents / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005609:Free Radicals / Q000737:chemistry* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000032:analysis; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D053719:Tandem Mass Spectrometry; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002254:Carbonates; D003432:Cross-Linking Reagents; D005609:Free Radicals; D014364:Tryptophan; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; C421864:hen egg lysozyme; D009113:Muramidase
keywords: Carbonate radical; Ditryptophan cross-link; Lysozyme dimer; Lysozyme oxidation; Lysozyme-superoxide dismutase dimer