Fetal hemoglobin is much less prone to DNA cleavage compared to the adult protein.
Fetal hemoglobin is much less prone to DNA cleavage compared to the adult protein.
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DOI：
10.1016/j.redox.2017.02.008
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发表时间：
2017-08
期刊：
影响因子：
11.4
通讯作者：
Bulow L
中科院分区：
文献类型：
--
作者：
Chakane S;Matos T;Kettisen K;Bulow L
血红蛋白（Hb）在红细胞（RBC）内受到很好的保护。在溶血时，当在循环中游离时，Hb可以参与一系列自由基生成反应，从而可以攻击几种不同的生物分子。在这项研究中，我们研究了潜在的破坏作用的无细胞血红蛋白的质粒DNA（pDNA）。Hb诱导超螺旋pDNA（sc pDNA）的裂解，其与施加的Hb浓度成比例。使用10 µM Hb在12 h内，几乎70%的sc pDNA转化为开环或线性DNA。Hb可以以几种不同的形式存在。氧（HbO2）和Met形式是最具反应性的，而羧基蛋白质仅显示出低水解活性。血红蛋白A（HbA）可以容易地诱导完全pDNA切割，而胎儿血红蛋白（HbF）的反应性低三倍。通过定点诱变在HbF α链表面插入氧化还原活性半胱氨酸残基，DNA切割反应增强了82%。活性氧不直接参与反应，因为加入超氧化物歧化酶和过氧化氢酶并不能阻止pDNA裂解。Hb与pDNA的反应性可能与基于蛋白质的自由基的形成有关。血红蛋白诱导质粒DNA切割在没有过氧化氢。胎儿血红蛋白的反应性比成人质粒DNA上的蛋白低三倍。在α链中插入半胱氨酸残基使DNA切割反应增强了82%。基于蛋白质的自由基与血红蛋白的DNA切割活性相关。
Hemoglobin (Hb) is well protected inside the red blood cells (RBCs). Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA). Hb induced cleavage of supercoiled pDNA (sc pDNA) which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10 µM of Hb in 12 h. Hb can be present in several different forms. The oxy (HbO2) and met forms are most reactive, while the carboxy-protein shows only low hydrolytic activity. Hemoglobin A (HbA) could easily induce complete pDNA cleavage while fetal hemoglobin (HbF) was three-fold less reactive. By inserting, a redox active cysteine residue on the surface of the alpha chain of HbF by site-directed mutagenesis, the DNA cleavage reaction was enhanced by 82%. Reactive oxygen species were not directly involved in the reaction since addition of superoxide dismutase and catalase did not prevent pDNA cleavage. The reactivity of Hb with pDNA can rather be associated with the formation of protein based radicals. Hemoglobin induced plasmid DNA cleavage in the absence of hydrogen peroxide. Fetal hemoglobin was three-fold less reactive compared to the adult protein on plasmid DNA. Insertion of a cysteine residue in the alpha chain enhanced the DNA cleavage reaction by 82%. Protein based radicals are associated with the DNA cleavage activity of hemoglobin.