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

Eduardo Fuentes-Lemus, Michele Mariotti, Per Hagglund, Fabian Leinisch, Angelica Fierro, Eduardo Silva, Camilo Lopez-Alarcon, Michael J. Davies

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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.
Original languageEnglish
JournalFree Radical Biology and Medicine
Volume143
Pages (from-to)375-386
ISSN0891-5849
DOIs
Publication statusPublished - 2019

Keywords

  • Rose bengal
  • Type 1 mechanism
  • Type 2 mechanism
  • Lysozyme
  • Protein cross-linking
  • Photo-oxidation
  • Tryptophan
  • Tyrosine

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