TY - JOUR

T1 - Binding of rose bengal to lysozyme modulates photooxidation and cross-linking reactions involving tyrosine and tryptophan

AU - Fuentes-Lemus, Eduardo

AU - Mariotti, Michele

AU - Hagglund, Per

AU - Leinisch, Fabian

AU - Fierro, Angelica

AU - Silva, Eduardo

AU - Lopez-Alarcon, Camilo

AU - Davies, Michael J.

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

KW - Rose bengal

KW - Type 1 mechanism

KW - Type 2 mechanism

KW - Lysozyme

KW - Protein cross-linking

KW - Photo-oxidation

KW - Tryptophan

KW - Tyrosine

U2 - 10.1016/j.freeradbiomed.2019.08.023

DO - 10.1016/j.freeradbiomed.2019.08.023

M3 - Journal article

C2 - 31446058

VL - 143

SP - 375

EP - 386

JO - Free Radical Biology & Medicine

JF - Free Radical Biology & Medicine

SN - 0891-5849

ER -