Browsing by Author "Mariotti, Michele"

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    Binding of rose bengal to lysozyme modulates photooxidation and cross-linking reactions involving tyrosine and tryptophan
    (2019) Fuentes Lemus, Eduardo Felipe; Mariotti, Michele; Hägglund, Per; Leinisch, Fabian; Fierro Huerta, Angélica; Silva, Eduardo; López Alarcón, Camilo Ignacio; Davies, Michael J.
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    Oxidation of lysozyme induced by peroxyl radicals involves amino acid modifications, loss of activity, and formation of specific crosslinks
    (2021) Fuentes Lemus, Eduardo Felipe; Mariotti, Michele; Hägglund, Per; Leinisch, Fabian; Fierro Huerta, Angélica; Silva Stevens, Eduardo Andrés; Davies, Michael J.; López Alarcón, Camilo Ignacio
    The 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.
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    Peroxyl radical- and photo-oxidation of glucose 6-phosphate dehydrogenase generates cross-links and functional changes via oxidation of tyrosine and tryptophan residues
    (2017) Leinisch, Fabian; Mariotti, Michele; Rykaer, Martin; López Alarcón, Camilo Ignacio; Davies, Michael J.; Hagglund, Per