P-46 - Influence of O2 on riboflavin-mediated photo-oxidation of lysozyme

Abstract

The oxidation of lysozyme (Lyso) mediated by photochemical agents has been previously studied, but the mechanisms involved are not fully understood. Lyso reacts with riboflavin triplet (3RF) involving tryptophan (Trp) oxidation with production of tryptophanyl radicals (Trp•). The later species react with oxygen (K > 10to5 M-1s-1) to form oxygenated compounds or self-react to generate di-Trp bonds. In the present work, we studied the influence of O2 on the 3RF-induced modifications of Lyso. Solutions containing Lyso (3 mg/mL) and riboflavin (35 µM) were illuminated with monochromatic visible light (450 nm) under N2, O2 (5%) and O2 (20%) environments. Spectroscopy, electrophoresis, and liquid chromatography with diode array and mass detection were employed as techniques. Lyso was efficiently oxidized by 3RF, Trp, Tyr and Met being the most oxidized residues. Under N2 atmosphere, monomers were consumed, but a low yield of dimers was observed. At O2 5%, a higher formation of dimers and trimers was detected together with formation of fragments. Interestingly, at O2 20%, the yield of Lyso crosslinks and fragments decreased. These results show a delicate balance between O2 concentration, the efficiency of riboflavin to generate 3RF and radical-radical reactions of Trp• (di-Trp bonds).