In this work we studied the mechanisms of Type I photodamage to a model protein, hen egg white lysozyme (HEWL), sensitized by kynurenic acid (KNA) - one of the most efficient photosensitizers of the human eye lens present in trace amounts within tissue. The kynurenic acid radical, KNA•-, formed in the quenching of triplet KNA by HEWL, can be readily oxidized by molecular oxygen with the formation of superoxide anion radical O2•-. This leads to two ways of damage to proteins: either via the direct reactions between KNA•- and HEWL• radicals (Type Ia) or via the reactions between superoxide anion O2•- and HEWL• radicals (Type Ib). Our results demonstrate significant degradation of the protein during Type Ia photolysis with the formation of various oligomeric and oxygenated forms of HEWL and several deoxygenated products of KNA. Liquid chromatography-mass spectrometry analysis revealed the cross-linking of HEWL via tryptophan (Trp62) and tyrosine (Tyr23) residues and, for the first time, the covalent binding of KNA to protein via tryptophan (Trp62 and Trp123) residues. It was found that Type Ib reactions lead to substantially smaller damage to HEWL; the degradation quantum yields (Φdeg) of HEWL are 1.3 ± 0.3% and 0.12 ± 0.03% for Type Ia and Ib photolyses, respectively. Low Φdeg values for both types of photolysis indicate the Back Electron Transfer (BET) with the restoration of initial reagents as the main radical decay path with significantly higher BET efficiency in the case of Type Ib reactions. Therefore, in essentially oxygen-free tissues like the eye lens, the direct radical reactions via Type Ia mechanism could induce significantly larger damage to proteins, leading to their cross-linking and oxidation. The accumulation of these modifications can cause the development of various diseases, in particular, cataracts in the eye lens.
|Журнал||Free radical biology & medicine|
|Состояние||Опубликовано - 20 мая 2020|