Induction of redox instability of bovine myoglobin by adduction with 4-hydroxy-2-nonenal
Induction of redox instability of bovine myoglobin by adduction with 4-hydroxy-2-nonenal
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DOI：
10.1021/bi0271695
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发表时间：
2003-04-22
期刊：
影响因子：
2.9
通讯作者：
Hill, DW
中科院分区：
文献类型：
--
作者：
Alderton, AL;Faustman, C;Hill, DW
肌红蛋白（Mb）的氧化还原稳定性受到许多因素的影响，包括脂质氧化及其产物。4-羟基-2-壬烯醛（HNE）是一种α，β-不饱和醛，来源于ω-6多不饱和脂肪酸的氧化，具有高度反应性和细胞毒性。我们的目的是研究HNE与Mb的潜在结合，并确定它如何影响氧化还原稳定性。将OxyMb（0.15 mM）与HNE（1 mM）在4、25和37 ℃下在pH 7.4或5.6下孵育。分析样品的MetMb形成，并通过蛋白质印迹分析、LC-MS、LC-MS-MS、圆二色性（CD）和差示扫描量热法（DSC）进行分析。MetMb的形成随温度升高而增加，在pH5.6时高于pH7.4时（P < 0.05）。在37 ℃时，HNE在pH7.4时加速氧化，而在pH5.6时不加速氧化（P < 0.05）。在25 ℃和4 ℃时，HNE对pH7.4和pH5.6的氧化均有促进作用（P < 0.05）。LC-MS显示HNE与Mb在两种pH值下通过Michael加成共价结合，而Western印迹分析表明HNE与组氨酸（HIS）残基结合。LC-MS-MS鉴定了Mb的六个组氨酸残基，其容易被HNE加合，包括与血红素基团相关的近端（HIS 93）和远端（HIS 64）组氨酸。CD未检测到对照Mb和与HNE孵育的Mb之间的二级结构差异。然而，DSC显示Mb与HNE在pH 7.4下反应的T-m降低，表明Mb三级结构以与去稳定化一致的方式改变。这些结果表明，HNE加速牛骨骼肌OxyMb在体外的组氨酸残基的共价修饰氧化。
The redox stability of myoglobin (Mb) is compromised by many factors, including lipid oxidation and its products. 4-Hydroxy-2-nonenal (HNE) is an alpha,beta-unsaturated aldehyde derived from the oxidation of omega-6 polyunsaturated fatty acids and is highly reactive and cytotoxic. Our objective was to study potential binding of HNE to Mb and determine how it affects redox stability. OxyMb (0.15 mM) was incubated with HNE (I mM) at 4, 25, and 37 degreesC at pH 7.4 or 5.6. Samples were analyzed for MetMb formation and by Western blot analyses, LC-MS, LC-MS-MS, circular dichroism (CD), and differential scanning calorimetry (DSC). MetMb formation increased with increasing temperature and was greater at pH 5.6 than at pH 7.4 (P < 0.05). At 37 degreesC, HNE accelerated oxidation at pH 7.4 but not at pH 5.6 (P < 0.05). At both 25 and 4 degreesC, HNE accelerated oxidation at pH 7.4 and 5.6 (P < 0.05). LC-MS revealed the covalent binding of HNE to Mb at both pH values via Michael addition, while Western blot analysis indicated that HNE was bound to histidine (HIS) residues. LC-MS-MS identified six histidine residues of Mb that were readily adducted by HNE, including the proximal (HIS 93) and distal (HIS 64) histidine associated with the heme group. Secondary structure differences between control Mb and Mb incubated with HNE were not detected by CD. However, DSC revealed a decreased T-m for Mb reacted with HNE at pH 7.4, indicating Mb tertiary structure was altered in a manner consistent with destabilization. These results suggest that HNE accelerates bovine skeletal muscle OxyMb oxidation in vitro by covalent modification at histidine residues.