TY - JOUR

T1 - Photo -oxidation of lysozyme triggered by riboflavin is O 2-dependent, occurs via mixed type 1 and type 2 pathways, and results in inactivation, site-specific damage and intra- and inter -molecular crosslinks

AU - Fuentes-Lemus, Eduardo

AU - Mariotti, Michele

AU - Reyes, Juan

AU - Leinisch, Fabian

AU - Hagglund, Per

AU - Silva, Eduardo

AU - Davies, Michael J.

AU - Lopez-Alarcon, Camilo

PY - 2020

Y1 - 2020

N2 - Photosensitized protein oxidation is a promising tool for medical procedures such as photochemical tissue bonding (PTB). We have recently reported that the binding of rose Bengal, a sensitizer employed in PTB, to lysozyme modulates the photooxidation and crosslinking of this protein. In this work we examined the photo - oxidation and crosslinking of lysozyme mediated by riboflavin (RF) an endogenous sensitizer also employed in PTB. We hypothesized that since RF does not bind strongly to proteins, the mechanism(s) and extent of enzy- matic inactivation, amino acid modification and protein crosslinking would be dependent on the presence of O2, and differ to that induced by rose Bengal. This hypothesis was tested using UV -visible spectrophotometry, isothermal titration calorimetry (ITC), SDS-PAGE gels, quantification of amino acid consumption, and LC -MS analysis of sites of modification and crosslinks. Under N2, limited damage was detected arising from type 1 (radical) chemistry with formation of specific intra- (Tyr20-Tyr23) and inter- (Tyr23-Trp108) molecular cross - links. In contrast, the presence of O2 triggered extensive protein damage through mixed type 1 and type 2 ( 1 O2) mechanisms leading to Trp, Met, Tyr and His oxidation, loss of enzymatic activity and protein dimerization. LC - MS analysis provided evidence for crosslinking via radical -radical recombination reactions (Trp28-Tyr53), and secondary reactions involving nucleophilic attack of the side -chain amine of Lys116 on carbonyl groups. Overall, this behavior is in marked contrast to that detected with rose Bengal indicating that the mechanisms and sites of photo -oxidative damage, and consequences for protein function, can be modulated by the choice of sensitizing dye.

AB - Photosensitized protein oxidation is a promising tool for medical procedures such as photochemical tissue bonding (PTB). We have recently reported that the binding of rose Bengal, a sensitizer employed in PTB, to lysozyme modulates the photooxidation and crosslinking of this protein. In this work we examined the photo - oxidation and crosslinking of lysozyme mediated by riboflavin (RF) an endogenous sensitizer also employed in PTB. We hypothesized that since RF does not bind strongly to proteins, the mechanism(s) and extent of enzy- matic inactivation, amino acid modification and protein crosslinking would be dependent on the presence of O2, and differ to that induced by rose Bengal. This hypothesis was tested using UV -visible spectrophotometry, isothermal titration calorimetry (ITC), SDS-PAGE gels, quantification of amino acid consumption, and LC -MS analysis of sites of modification and crosslinks. Under N2, limited damage was detected arising from type 1 (radical) chemistry with formation of specific intra- (Tyr20-Tyr23) and inter- (Tyr23-Trp108) molecular cross - links. In contrast, the presence of O2 triggered extensive protein damage through mixed type 1 and type 2 ( 1 O2) mechanisms leading to Trp, Met, Tyr and His oxidation, loss of enzymatic activity and protein dimerization. LC - MS analysis provided evidence for crosslinking via radical -radical recombination reactions (Trp28-Tyr53), and secondary reactions involving nucleophilic attack of the side -chain amine of Lys116 on carbonyl groups. Overall, this behavior is in marked contrast to that detected with rose Bengal indicating that the mechanisms and sites of photo -oxidative damage, and consequences for protein function, can be modulated by the choice of sensitizing dye.

KW - Riboflavin

KW - Type 1 mechanism

KW - Type 2 mechanism

KW - Lysozyme

KW - Protein crosslinking

KW - Photo-oxidation

KW - Tryptophan

KW - Tyrosine

U2 - 10.1016/j.freeradbiomed.2020.03.004

DO - 10.1016/j.freeradbiomed.2020.03.004

M3 - Journal article

C2 - 32142879

VL - 152

SP - 61

EP - 73

JO - Free Radical Biology & Medicine

JF - Free Radical Biology & Medicine

SN - 0891-5849

ER -