Cohen G, Yakushin S, Dembiec-Cohen D
Department of Neurology and Neurobiology Research Center, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029, USA.
Anal Biochem. 1998 Oct 15;263(2):232-9. doi: 10.1006/abio.1998.2766.
It is widely believed that hydroxyl radicals generated in vivo contribute to damage to macromolecules, such as proteins and DNA. We evaluated methodology based on the transformation of protein tyrosine to L-Dopa, via aromatic ring hydroxylation, as an index of hydroxyl radical attack on proteins. The catechol structure of Dopa makes it amenable to isolation with alumina, followed by HPLC analysis, typically used for the measurement of catecholamines. Because a level of controversy exists about the formation of Dopa by hydroxyl radicals, we conducted a systematic study of the formation of Dopa from tyrosine, tyrosine dipeptides, pure proteins (chymotrypsin and myelin basic protein), and endogenous proteins in tissue homogenates (rat brain), exposed to hydroxylating conditions (Fe2+/EDTA/ascorbate at neutral pH). Dopa residues in peptides and proteins were liberated by acid hydrolysis with 6 M HCl at 145 degrees C for 1 h. A marked lability of Dopa in 6 M HCl under hydrolysis conditions was prevented with added phenol; chymotrypsin and precipitated pellets of brain protein were also protective. Overall recoveries (hydrolysis plus purification procedures) averaged 83.4 +/- 1.7%. This improved analytic procedure may be useful for studying protein damage by hydroxyl radicals.
人们普遍认为，体内产生的羟基自由基会对蛋白质和DNA等大分子造成损伤。我们评估了一种方法，该方法基于通过芳香环羟基化将蛋白质酪氨酸转化为L-多巴，以此作为羟基自由基对蛋白质攻击的指标。多巴的儿茶酚结构使其适合用氧化铝分离，随后进行HPLC分析，这是通常用于测定儿茶酚胺的方法。由于关于羟基自由基形成多巴存在一定争议，我们对在羟基化条件下（中性pH值的Fe2+/EDTA/抗坏血酸），酪氨酸、酪氨酸二肽、纯蛋白质（胰凝乳蛋白酶和髓鞘碱性蛋白）以及组织匀浆（大鼠脑）中的内源性蛋白质形成多巴的情况进行了系统研究。肽和蛋白质中的多巴残基通过在145℃下用6 M HCl酸水解1小时而释放出来。添加苯酚可防止多巴在水解条件下于6 M HCl中显著不稳定；胰凝乳蛋白酶和脑蛋白沉淀颗粒也具有保护作用。总体回收率（水解加纯化程序）平均为83.4±1.7%。这种改进的分析方法可能有助于研究羟基自由基对蛋白质的损伤。
