Oxidation of lysozyme induced by peroxyl radicals involves amino acid modifications, loss of activity, and formation of specific crosslinks

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dc.catalogadorgjm
dc.contributor.authorFuentes Lemus, Eduardo Felipe
dc.contributor.authorMariotti, Michele
dc.contributor.authorHägglund, Per
dc.contributor.authorLeinisch, Fabian
dc.contributor.authorFierro Huerta, Angélica
dc.contributor.authorSilva Stevens, Eduardo Andrés
dc.contributor.authorDavies, Michael J.
dc.contributor.authorLópez Alarcón, Camilo Ignacio
dc.date.accessioned2024-12-30T19:02:06Z
dc.date.available2024-12-30T19:02:06Z
dc.date.issued2021
dc.description.abstractThe present work examined the oxidation and crosslinking of the anti-bacterial enzyme lysozyme (Lyso), which is present in multiple biological fluids, and released from the cytoplasmic granules of macrophages and neutrophils at sites of infection and inflammation. It is therefore widely exposed to oxidants including peroxyl radicals (ROO?). We hypothesized that exposure to ROO? would generate specific modifications and inter- and intraprotein crosslinks via radical-radical reactions. Lyso was incubated with AAPH (2,2?-azobis(2-methylpropionamidine) dihydrochloride) as a ROO? source. Enzymatic activity was assessed, while oxidative modifications were detected and quantified using electrophoresis and liquid chromatography (UPLC) with fluorescence or mass detection (MS). Computational models of AAPH-Lyso interactions were developed. Exposure of Lyso to AAPH (10 and 100 mM for 3 h, and 20 mM for 1 h), at 37 ?C, decreased enzymatic activity. 20 mM AAPH showed the highest efficiency of Lyso inactivation (1.78 mol of Lyso inactivated per ROO?). Conversion of Met to its sulfoxide, and to a lesser extent, Tyr oxidation to 3,4-dihydroxyphenylalanine and diTyr, were detected by UPLCMS. Extensive transformation of Trp, involving short chain reactions, to kynurenine, oxindole, hydroxytryptophan, hydroperoxides or di-alcohols, and N-formyl-kynurenine was detected, with Trp62, Trp63 and Trp108 the most affected residues. Interactions of AAPH inside the negatively-charged catalytic pocket of Lyso, with Trp108, Asp52, and Glu35, suggest that Trp108 oxidation mediates, at least partly, Lyso inactivation. Crosslinks between Tyr20-Tyr23 (intra-molecular), and Trp62-Tyr23 (inter-molecular), were detected with both proximity (Tyr20-Tyr23), and chain flexibility (Trp62) appearing to favor the formation of covalent crosslinks.
dc.format.extent13 páginas
dc.fuente.origenConveris
dc.identifier.doi10.1016/j.freeradbiomed.2021.03.009
dc.identifier.issn0891-5849
dc.identifier.scopusidSCOPUS_ID:2-s2.0-85103312655
dc.identifier.urihttps://doi.org/10.1016/j.freeradbiomed.2021.03.009
dc.identifier.urihttps://repositorio.uc.cl/handle/11534/89385
dc.identifier.wosidWOS:000646309500003
dc.information.autorucEscuela de Química; Fuentes Lemus, Eduardo Felipe; 0000-0002-1465-8466; 186720
dc.information.autorucEscuela de Química; Fierro Huerta, Angélica; 0000-0002-6507-4188; 218137
dc.information.autorucEscuela de Química; Silva Stevens, Eduardo Andrés; S/I; 98771
dc.information.autorucEscuela de Química; López Alarcón, Camilo Ignacio; S/I; 1004308
dc.language.isoen
dc.nota.accesocontenido parcial
dc.pagina.final270
dc.pagina.inicio258
dc.revistaFree Radical Biology and Medicine
dc.rightsacceso restringido
dc.subjectLysozyme
dc.subjectPeroxyl radicals
dc.subjectProtein crosslinking
dc.subjectEnzymatic activity
dc.subjectTryptophan oxidation
dc.subjectRadical-radical reactions
dc.subject.ddc510
dc.subject.deweyMatemática física y químicaes_ES
dc.titleOxidation of lysozyme induced by peroxyl radicals involves amino acid modifications, loss of activity, and formation of specific crosslinks
dc.typeartículo
dc.volumen167
sipa.codpersvinculados186720
sipa.codpersvinculados218137
sipa.codpersvinculados98771
sipa.codpersvinculados1004308
sipa.trazabilidadConveris;20-07-2021
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