Davies K J
J Biol Chem. 1987 Jul 15;262(20):9895-901.
Aggregation, fragmentation, amino acid modification, and proteolytic susceptibility have been studied following exposure of 17 proteins to oxygen radicals. The hydroxyl radical (.OH) produced covalently bound protein aggregates, but few or no fragmentation products. Extensive changes in net electrical charge (both + and -) were observed. Tryptophan was rapidly lost with .OH exposure, and significant production of bityrosine biphenol occurred. When incubated with cell-free extracts of human and rabbit erythrocytes, rabbit reticulocytes, or Escherichia coli, most .OH-modified proteins were proteolytically degraded up to 50 times faster than untreated proteins. The exceptions were alpha-casein and globin, which were rapidly degraded without .OH modification. ATP did not stimulate the degradation of .OH-modified proteins, but alpha-casein was more rapidly degraded. Leupeptin had little effect under any condition, and degradation was maximal at pH 7.8. The data indicate that proteins which have been denatured by .OH can be recognized and degraded rapidly and selectively by intracellular proteolytic systems. In both red blood cells and E. coli, the degradation appears to be conducted by soluble, ATP-independent (nonlysosomal) proteolytic enzymes. In contrast with the above results, superoxide (O2-) did not cause aggregation or fragmentation, tryptophan loss, or bityrosine production. The combination of .OH + O2- (+O2), which may mimic biological exposure to oxygen radicals, induced charge changes, tryptophan loss, and bityrosine production. The pattern of such changes was similar to that seen with .OH alone, although the extent was generally less severe. In contrast with .OH alone, however, .OH + O2- (+O2) caused extensive protein fragmentation and little or no aggregation. More than 98% of the protein fragments had molecular weights greater than 5000 and formed clusters of ionic and hydrophobic bonds which could be dispersed by denaturing agents. The results indicate a general sensitivity of proteins to oxygen radicals. Oxidative modification can involve direct fragmentation or may provide denatured substrates for intracellular proteolysis.
在使17种蛋白质暴露于氧自由基后，对其聚集、碎片化、氨基酸修饰和蛋白水解敏感性进行了研究。产生的羟基自由基（·OH）形成了共价结合的蛋白质聚集体，但几乎没有或没有碎片化产物。观察到净电荷发生了广泛变化（包括正电荷和负电荷）。色氨酸在暴露于·OH时迅速损失，并且大量生成了二酪氨酸二酚。当与人和兔红细胞、兔网织红细胞或大肠杆菌的无细胞提取物一起孵育时，大多数经·OH修饰的蛋白质被蛋白水解降解的速度比未处理的蛋白质快50倍。例外情况是α-酪蛋白和球蛋白，它们在未经·OH修饰的情况下也会迅速降解。ATP不会刺激经·OH修饰的蛋白质的降解，但α-酪蛋白降解得更快。亮抑酶肽在任何条件下影响都很小，并且在pH 7.8时降解达到最大值。数据表明，被·OH变性的蛋白质可以被细胞内蛋白水解系统快速且选择性地识别和降解。在红细胞和大肠杆菌中，降解似乎是由可溶性的、不依赖ATP（非溶酶体）的蛋白水解酶进行的。与上述结果相反，超氧阴离子（O2-）不会引起聚集或碎片化、色氨酸损失或二酪氨酸生成。·OH + O2-（+O2）的组合可能模拟生物对氧自由基的暴露，诱导了电荷变化、色氨酸损失和二酪氨酸生成。这种变化模式与单独使用·OH时相似，尽管程度通常较轻。然而，与单独的·OH相比，·OH + O2-（+O2）导致了广泛的蛋白质碎片化，几乎没有或没有聚集。超过98%的蛋白质片段分子量大于5000，并形成了离子键和疏水键簇，这些键簇可被变性剂分散。结果表明蛋白质对氧自由基普遍敏感。氧化修饰可能涉及直接碎片化，也可能为细胞内蛋白水解提供变性底物。
