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

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 - 2019/11/1

Y1 - 2019/11/1

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 1O2 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 H2 18O 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 Kd of 4.8 μM, while data from ITC studies, yielded a Kd of 0.68 μM 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 (1O2) 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 1O2 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 H2 18O 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 Kd of 4.8 μM, while data from ITC studies, yielded a Kd of 0.68 μM 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 (1O2) 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

SN - 0891-5849

VL - 143

SP - 375

EP - 386

JO - Free Radical Biology & Medicine

JF - Free Radical Biology & Medicine

ER -