Protein damage and degradation by oxygen radicals. III. Modification of secondary and tertiary structure.
Protein damage and degradation by oxygen radicals. III. Modification of secondary and tertiary structure.
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DOI：
10.1016/s0021-9258(18)48020-9
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发表时间：
1987-07
期刊：
影响因子：
--
通讯作者：
K. Davies;M. Delsignore
中科院分区：
文献类型：
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作者：
K. Davies;M. Delsignore
暴露于羟基自由基（.OH）或.OH+超氧阴离子自由基和氧的组合（.OH + O2- + O2）的蛋白质表现出改变的一级结构和增加的蛋白水解敏感性。目前的工作表明，一级结构的改变导致二级和三级结构的严重扭曲。.OH或.OH + O2- + O2对牛血清白蛋白（BSA）的变性/疏水性增加在自由基/BSA摩尔比为24（全部.OH或50%.OH +50%O2-）时最大。暴露于.OH的BSA也经历了渐进的共价交联，形成二聚体、三聚体和四聚体，部分原因是形成了分子间的酪氨酸。OH + O2- + O2引起BSA自发断裂。BSA片段化产生新的羰基，而游离氨基没有明显增加。断裂可能涉及（. OH诱导的）α-碳自由基与O2反应形成过氧自由基，过氧自由基分解在α-碳而不是在肽键处使多肽链断裂。在变性条件下电泳后，OH + O2- + O2诱导的BSA片段显示分子量为7，000 - 60，000，但在非变性凝胶中可观察到疏水聚集体（用尿素或酸处理后用[3 H]BSA证实）。各种化学自由基清除剂（甘露醇，尿酸盐，叔丁醇，异丙醇）和气体（N2 O，O2，N2）的组合显示，.OH是负责改变BSA的二级和三级结构的主要物种。氧和O2-仅用于改变.OH反应的结果。此外，O2- + O2（在没有.OH的情况下）的直接研究显示BSA结构没有可测量的变化。发现变性/疏水性增加的过程先于共价交联（通过.OH）或断裂（通过.OH + O2- + O2）。变性是半最大的自由基/BSA的摩尔比为9.6，而半最大的聚集或碎片发生在19.4的比例。变性/疏水性可能为氧自由基增加蛋白水解敏感性的机制提供重要线索。
Proteins which have been exposed to the hydroxyl radical (.OH) or to the combination of .OH plus the superoxide anion radical and oxygen (.OH + O2- + O2) exhibit altered primary structure and increased proteolytic susceptibility. The present work reveals that alterations to primary structure result in gross distortions of secondary and tertiary structure. Denaturation/increased hydrophobicity of bovine serum albumin (BSA) by .OH, or by .OH + O2- + O2 was maximal at a radical/BSA molar ratio of 24 (all .OH or 50% .OH + 50% O2-). BSA exposed to .OH also underwent progressive covalent cross-linking to form dimers, trimers, and tetramers, partially due to the formation of intermolecular bityrosine. In contrast, .OH + O2- + O2 caused spontaneous BSA fragmentation. Fragmentation of BSA produced new carbonyl groups with no apparent increase in free amino groups. Fragmentation may involve reaction of (.OH-induced) alpha-carbon radicals with O2 to form peroxyl radicals which decompose to fragment the polypeptide chain at the alpha-carbon, rather than at peptide bonds. BSA fragments induced by .OH + O2- + O2 exhibited molecular weights of 7,000-60,000 following electrophoresis under denaturing conditions, but could be visualized as hydrophobic aggregates in nondenaturing gels (confirmed with [3H]BSA following treatment with urea or acid). Combinations of various chemical radical scavengers (mannitol, urate, t-butyl alcohol, isopropyl alcohol) and gases (N2O, O2, N2) revealed that .OH is the primary species responsible for alteration of BSA secondary and tertiary structure. Oxygen, and O2- serve only to modify the outcome of .OH reaction. Furthermore, direct studies of O2- + O2 (in the absence of .OH) revealed no measurable changes in BSA structure. The process of denaturation/increased hydrophobicity was found to precede either covalent cross-linking (by .OH) or fragmentation (by .OH + O2- + O2). Denaturation was half-maximal at a radical/BSA molar ratio of 9.6, whereas half-maximal aggregation or fragmentation occurred at a ratio of 19.4. Denaturation/hydrophobicity may hold important clues for the mechanism(s) by which oxygen radicals can increase proteolytic susceptibility.