Escuela de Nutrición y Dietética, Facultad de Ciencias de la Salud, Universidad de Talca, Talca, Chile; Interdisciplinary Center on Aging, Universidad de Talca, Talca, Chile.
Escuela de Nutrición y Dietética, Facultad de Ciencias de la Salud, Universidad de Talca, Talca, Chile; Interdisciplinary Center on Aging, Universidad de Talca, Talca, Chile.
Free Radic Biol Med. 2019 Sep;141:103-114. doi: 10.1016/j.freeradbiomed.2019.05.024. Epub 2019 May 22.
Photosensitized reactions mediated by endogenous chromophores have been associated with the etiology of age-related cataract disease. Endogenous chromophores such as 3-hydroxykynurenine (3OHKN) can be found in both free form, and bound to crystallin proteins. However, their efficiency in generating photo-induced oxidative modifications on eye lens proteins is not completely understood. In this work, the efficiency and photodynamic activity of 3OHKN bound to both lysine (3OHKN-Lys) and bovine lens proteins (3OHKN-BLP) was assessed and compared with the photosensitizing activity of the major chromophore arising from glucose degradation (GDC). The photosensitizing activity of 3OHKN-Lys, 3OHKN-BLP and GDC was characterized by measurement of singlet oxygen quantum yields, O consumption, SDS-PAGE and amino acid analysis of the photo-oxidized proteins. Singlet oxygen quantum yields under 20% O atmosphere were 0.02, 0.01, and 0.27 for 3OHKN-Lys, 3OHKN-BLP and GDC, respectively. O consumption by photosensitized reactions was more efficient for 3OHKN-BLP, with the extent of O consumption being ∼28% higher than for 3OHKN-Lys and GDC under both 5 and 20% O. SDS-PAGE showed that protein crosslinking is dependent on the O concentration, and more extensive at 5 than 20% O. GDC and 3OHKN-Lys were the most efficient crosslinkers at 20 and 5% O, respectively. Amino acid analysis of the irradiated proteins showed consumption of Trp, His, Tyr and Phe, and formation of kynurenine (from Trp), methionine sulfoxide (from Met) and DOPA (from Tyr). Kynurenine formation was dependent on the O concentration with higher amounts detected at 5 than 20% O for 3OHKN-BLP and 3OHKN-Lys, with 3OHKN-BLP the most efficient sensitizer. Our results suggest that 3OHKN-BLP can elicit photo-oxidative damage mainly by a type I photosensitizing mechanism, with this likely to be the most prevalent pathway at the low physiologic O concentrations in the eye lens.
内源性发色团介导的光致敏反应与年龄相关性白内障疾病的病因有关。内源性发色团，如 3-羟基犬尿氨酸（3OHKN），可以以游离形式和与晶体蛋白结合的形式存在。然而，它们在生成眼晶状体蛋白光诱导氧化修饰方面的效率尚不完全清楚。在这项工作中，评估了 3OHKN 与赖氨酸（3OHKN-Lys）和牛晶状体蛋白（3OHKN-BLP）结合的效率和光动力活性，并与葡萄糖降解产生的主要发色团（GDC）的光致敏活性进行了比较。通过测量单线态氧量子产率、O 消耗、光氧化蛋白的 SDS-PAGE 和氨基酸分析来表征 3OHKN-Lys、3OHKN-BLP 和 GDC 的光致敏活性。在 20%O 气氛下，3OHKN-Lys、3OHKN-BLP 和 GDC 的单线态氧量子产率分别为 0.02、0.01 和 0.27。光致敏反应的 O 消耗效率对 3OHKN-BLP 更高，在 5%和 20%O 下，O 消耗的程度比 3OHKN-Lys 和 GDC 分别高约 28%。SDS-PAGE 显示蛋白质交联依赖于 O 浓度，在 5%O 下比 20%O 更广泛。GDC 和 3OHKN-Lys 分别是 20%和 5%O 下最有效的交联剂。对辐照蛋白的氨基酸分析表明色氨酸、组氨酸、酪氨酸和苯丙氨酸的消耗以及犬尿氨酸（来自色氨酸）、蛋氨酸亚砜（来自蛋氨酸）和多巴（来自酪氨酸）的形成。犬尿氨酸的形成依赖于 O 浓度，在 5%O 下比 20%O 下检测到的 3OHKN-BLP 和 3OHKN-Lys 更多，3OHKN-BLP 是最有效的敏化剂。我们的结果表明，3OHKN-BLP 可以主要通过 I 型光致敏机制引起光氧化损伤，这可能是眼晶状体中低生理 O 浓度下最常见的途径。
