Binding of rose bengal to lysozyme modulates photooxidation and cross-linking reactions involving tyrosine and tryptophan

Fuentes-Lemus, Eduardo; Mariotti, Michele; Hägglund, Per; Leinisch, Fabian; Fierro, Angélica; Silva, Eduardo; López-Alarcón, Camilo; Davies, Michael J.

Abstract

This work examined the hypothesis that interactions of Rose Bengal (RB2-) with lysozyme (Lyso) might mediate type 1 photoreactions resulting in protein cross-linking even under conditions favoring O-1(2) formation. UV-visible spectrophotometry, isothermal titration calorimetry (ITC), and docking analysis were employed to characterize RB2--Lyso interactions, while oxidation of Lyso was studied by SDS-PAGE gels, extent of amino acid consumption, and liquid chromatography (LC) with mass detection (employing tryptic peptides digested in H-2 O-18 and H2O). Docking studies showed five interaction sites including the active site. Hydrophobic interactions induced a red shift of the visible spectrum of RB2- giving a K-d of 4.8 mu M, while data from ITC studies, yielded a K-d of 0.68 mu M as an average of the interactions with stoichiometry of 3.3 RB2- per Lyso. LC analysis showed a high consumption of readily-oxidized amino acids (His, Trp, Met and Tyr) located at different and diverse locations within the protein. This appears to reflect extensive damage on the protein probably mediated by a type 2 (O-1(2)) mechanism. In contrast, docking and mass spectrometry analysis provided evidence for the generation of specific intra- (Tyr23-Tyr20) and inter-molecular (Tyr23-Trp62) Lyso cross-links, and Lyso dimer formation via radical-radical, type 1 mechanisms.

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Título según WOS: Binding of rose bengal to lysozyme modulates photooxidation and cross-linking reactions involving tyrosine and tryptophan
Título según SCOPUS: Binding of rose bengal to lysozyme modulates photooxidation and cross-linking reactions involving tyrosine and tryptophan
Título de la Revista: FREE RADICAL BIOLOGY AND MEDICINE
Volumen: 143
Editorial: Elsevier Science Inc.
Fecha de publicación: 2019
Página de inicio: 375
Página final: 386
Idioma: English
DOI:

10.1016/j.freeradbiomed.2019.08.023

Notas: ISI, SCOPUS - ISI, SCOPUS