Savina Ekaterina D, Tsentalovich Yuri P, Sherin Peter S
International Tomography Center SB RAS, Institutskaya str. 3A, 630090, Novosibirsk, Russia; Novosibirsk State University, Pirogova str. 2, 630090, Novosibirsk, Russia.
International Tomography Center SB RAS, Institutskaya str. 3A, 630090, Novosibirsk, Russia; Novosibirsk State University, Pirogova str. 2, 630090, Novosibirsk, Russia.
Free Radic Biol Med. 2020 May 20;152:482-493. doi: 10.1016/j.freeradbiomed.2019.11.017. Epub 2019 Nov 18.
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 O. 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 O 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 (Trp) and tyrosine (Tyr) residues and, for the first time, the covalent binding of KNA to protein via tryptophan (Trp and Trp) residues. It was found that Type Ib reactions lead to substantially smaller damage to HEWL; the degradation quantum yields (Φ) of HEWL are 1.3 ± 0.3% and 0.12 ± 0.03% for Type Ia and Ib photolyses, respectively. Low Φ 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.
在这项工作中，我们研究了犬尿氨酸（KNA）敏化下模型蛋白——鸡蛋清溶菌酶（HEWL）的I型光损伤机制。KNA是人眼晶状体中含量极少但效率极高的光敏剂之一。HEWL猝灭三重态KNA时形成的犬尿氨酸自由基KNA可被分子氧轻易氧化，生成超氧阴离子自由基O。这导致蛋白质损伤有两种方式：要么通过KNA与HEWL自由基之间的直接反应（Ia型），要么通过超氧阴离子O与HEWL自由基之间的反应（Ib型）。我们的结果表明，在Ia型光解过程中蛋白质发生了显著降解，形成了各种寡聚和氧化形式的HEWL以及几种KNA的脱氧产物。液相色谱 - 质谱分析揭示了HEWL通过色氨酸（Trp）和酪氨酸（Tyr）残基发生交联，并且首次发现KNA通过色氨酸（Trp和Trp）残基与蛋白质发生共价结合。研究发现，Ib型反应对HEWL的损伤要小得多；Ia型和Ib型光解中HEWL的降解量子产率（Φ）分别为1.3±0.3%和0.12±0.03%。两种光解类型的低Φ值表明存在反向电子转移（BET），初始试剂的恢复是主要的自由基衰变途径，在Ib型反应中BET效率显著更高。因此，在像眼晶状体这样基本无氧的组织中，通过Ia型机制的直接自由基反应可能会对蛋白质造成显著更大的损伤，导致其交联和氧化。这些修饰的积累可能会引发各种疾病，特别是眼晶状体中的白内障。
