Morin B, Davies M J, Dean R T
Cell Biology Unit, The Heart Research Institute, 145-147 Missenden Road, Camperdown, Sydney, NSW2050, Australia.
Biochem J. 1998 Mar 15;330 ( Pt 3)(Pt 3):1059-67. doi: 10.1042/bj3301059.
A major product of hydroxy-radical addition to tyrosine is 3, 4-dihydroxyphenylalanine (DOPA) which has reducing properties. Protein-bound DOPA (PB-DOPA) has been shown to be a major component of the stable reducing species formed during protein oxidation under several conditions. The aim of the present work was to investigate whether DOPA, and especially PB-DOPA, can mediate oxidative damage to DNA. We chose to generate PB-DOPA using mushroom tyrosinase, which catalyses the hydroxylation of tyrosine residues in protein. This permitted us to study the reactions of PB-DOPA in the virtual absence of other protein-bound oxidation products. The formation of two oxidation products of DNA, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG) and 5-hydroxy-2'-deoxycytidine (5OHdC), were studied with a novel HPLC using gradient elution and an electrochemical detection method, which allowed the detection of both DNA modifications in a single experiment. We found that exposure of calf thymus DNA to DOPA or PB-DOPA resulted in the formation of 8oxodG and 5OHdC, with the former predominating. The formation of these DNA oxidation products by either DOPA or PB-DOPA depended on the presence of oxygen, and also on the presence and on the concentration of transition metal ions, with copper being more effective than iron. The yields of 8oxodG and 5OHdC increased with DOPA concentration in proteins. Thus PB-DOPA was able to promote further radical-generating events, which then transferred damage to other biomolecules such as DNA.
羟基自由基加成到酪氨酸上的一个主要产物是具有还原性质的3,4-二羟基苯丙氨酸（多巴）。在几种条件下，蛋白质结合的多巴（PB - 多巴）已被证明是蛋白质氧化过程中形成的稳定还原物质的主要成分。本研究的目的是调查多巴，尤其是PB - 多巴，是否能介导对DNA的氧化损伤。我们选择使用蘑菇酪氨酸酶生成PB - 多巴，该酶催化蛋白质中酪氨酸残基的羟基化。这使我们能够在几乎不存在其他蛋白质结合氧化产物的情况下研究PB - 多巴的反应。使用新型高效液相色谱（HPLC）梯度洗脱和电化学检测方法研究了DNA的两种氧化产物8-氧代-7,8-二氢-2'-脱氧鸟苷（8-氧代脱氧鸟苷，8oxodG）和5-羟基-2'-脱氧胞苷（5-羟基脱氧胞苷，5OHdC） 的形成，该方法允许在单个实验中检测这两种DNA修饰。我们发现，小牛胸腺DNA暴露于多巴或PB - 多巴会导致8oxodG和5OHdC的形成，前者占主导。多巴或PB - 多巴形成这些DNA氧化产物取决于氧气的存在，也取决于过渡金属离子的存在和浓度，其中铜比铁更有效。8oxodG和5OHdC的产量随着蛋白质中多巴浓度的增加而增加。因此，PB - 多巴能够促进进一步的自由基生成事件，进而将损伤转移到其他生物分子如DNA上。
