Nitrogen dioxide reactivity with proteins: effects on activity and immunoreactivity with alpha-1-proteinase inhibitor and implications for NO2-mediated peptide degradation.
Nitrogen dioxide reactivity with proteins: effects on activity and immunoreactivity with alpha-1-proteinase inhibitor and implications for NO2-mediated peptide degradation.
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二氧化氮与蛋白质的反应性：对 α-1-蛋白酶抑制剂的活性和免疫反应性的影响以及对 NO2 介导的肽降解的影响。
DOI：
10.1006/abbi.1993.1316
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发表时间：
1993
影响因子：
3.9
通讯作者：
Johnson,DA
中科院分区：
文献类型：
--
作者：
Hood,DB;Gettins,P;Johnson,DA
二氧化氮（NO2）是燃烧化石燃料产生的一种空气污染物，也是香烟烟雾的一种成分，被认为是肺气肿等肺部疾病的发病机制。为了获得有关NO2损害肺部和对其功能至关重要的蛋白质的机制以及其与一般蛋白质的反应的信息，进行了α-1-蛋白酶抑制剂（α 1 PI）、弹性蛋白、聚-L-赖氨酸和聚-L-精氨酸的体外暴露。当NO：α 12 PI的摩尔比为400：1或更高时，α 1 PI抑制其天然生理靶点人中性粒细胞弹性蛋白酶（HNE）的能力随着暴露于对照值的54%而下降。在酶联免疫吸附试验中，当NO：α 12 PI的摩尔比为100：1或更大时，α 1 PI暴露于NO2导致单克隆或多克隆抗体的免疫反应性损失50%。邻苯二甲醛和二辛可宁酸蛋白质平行测定以及对照和NO2暴露的α 1 PI的氨基酸分析结果表明NO2与赖氨酸残基反应。弹性蛋白和多聚-L-赖氨酸在生理pH的水溶液中用[3 H]HCHO还原甲基化氨基，然后用NO2处理。NO2暴露弹性蛋白导致84%的相关放射性溶解，其中79%通过氨基酸分析鉴定为[3 H]甲基赖氨酸。聚L-[~ 3 H]赖氨酸经NO_2暴露后，凝胶过滤层析显示50，000 Mr的聚L-[~ 3 H]赖氨酸已被降解为1- 3000 Mr的小肽。类似地，未标记的聚-L-精氨酸暴露于NO2后，凝胶过滤色谱和总肽分析显示47，500 Mr肽也部分降解为肽。这些结果表明，NO2与赖氨酸残基的α-氨基（伯胺）和与表面暴露的赖氨酸和精氨酸残基的酰胺氮（仲胺）在肽骨架中的反应，导致肽键断裂。这些发现是NO2介导的肽降解的第一个迹象，并提供了额外的数据，NO2的潜在损害蛋白质至关重要的肺在体外暴露系统的功能。
Nitrogen dioxide (NO2), an air pollutant produced by burning fossil fuels and a component of cigarette smoke, is thought to contribute to the pathogenesis of pulmonary diseases, such as emphysema. In order to gain information on the mechanism by which NO2damages the lung and proteins vital to its function, as well as its reaction with proteins in general,in vitroexposures of α-1-proteinase inhibitor (α1PI), elastin, poly-L-lysine, and poly-L-arginine were performed. The ability of α1PI to inhibit its natural physiological target, human neutrophil elastase (HNE), declined with exposure to 54% of the control value at molar ratios of NO:α12PI of 400:1 and greater. Exposure of α1PI to NO2resulted in a 50% loss of immunoreactivity with either monoclonal or polyclonal antibodies in an enzyme-linked immunosorbent assay at molar ratios of NO:α12PI of 100:1 and greater. The results of parallelO-phthalaldehyde and bicinchoninic acid protein assays as well as amino acid analysis on control and NO2-exposed α1PI suggested a reactivity of NO2with lysine residues. Elastin and poly-L-lysine were labeled by reductive methylation of amino groups with [3H]HCHO prior to treatment with NO2in aqueous solutions at physiological pH. NO2exposure of elastin resulted in the solubilization of 84% of the associated radioactivity of which 79% was identified as [3H]methyllysine by amino acid analysis. After NO2exposure of poly-L-[3H]lysine, gel filtration chromatography revealed that the 50,000Mrpoly-L-[3H]lysine had been degraded to small peptides of 1-3000Mr. Similarly, after NO2exposure of unlabeled poly-L-arginine, gel filtration chromatography, and total peptide analysis revealed that the 47,500Mrpeptide was also partially degraded to peptides. These results suggest that NO2reacts with the ϵ-amino groups of Lys residues (primary amines) and with the amide nitrogen (secondary amines) of surface-exposed Lys and Arg residues in the peptide backbone to result in peptide bond cleavage. These findings are the first indication of NO2-mediated peptide degradation and provide additional data on the potential of NO2to damage proteins vital to the function of the lung in anin vitroexposure system.