Hood D B, Gettins P, Johnson D A
Department of Biochemistry, James H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614-0581.
Arch Biochem Biophys. 1993 Jul;304(1):17-26. doi: 10.1006/abbi.1993.1316.
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 NO2 damages the lung and proteins vital to its function, as well as its reaction with proteins in general, in vitro exposures of alpha-1-proteinase inhibitor (alpha 1PI), elastin, poly-L-lysine, and poly-L-arginine were performed. The ability of alpha 1PI to inhibit its natural physiological target, human neutrophil elastase (HNE), declined with exposure to 54% of the control value at molar ratios of NO2:alpha 1PI of 400:1 and greater. Exposure of alpha 1PI to NO2 resulted in a 50% loss of immunoreactivity with either monoclonal or polyclonal antibodies in an enzyme-linked immunosorbent assay at molar ratios of NO2:alpha 1PI of 100:1 and greater. The results of parallel O-phthalaldehyde and bicinchoninic acid protein assays as well as amino acid analysis on control and NO2-exposed alpha 1PI suggested a reactivity of NO2 with lysine residues. Elastin and poly-L-lysine were labeled by reductive methylation of amino groups with [3H]HCHO prior to treatment with NO2 in aqueous solutions at physiological pH. NO2 exposure 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 NO2 exposure of poly-L-[3H]lysine, gel filtration chromatography revealed that the 50,000 M(r) poly-L-[3H]lysine had been degraded to small peptides of 1-3000 M(r). Similarly, after NO2 exposure of unlabeled poly-L-arginine, gel filtration chromatography, and total peptide analysis revealed that the 47,500 M(r) peptide was also partially degraded to peptides. These results suggest that NO2 reacts with the epsilon-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 NO2 to damage proteins vital to the function of the lung in an in vitro exposure system.
二氧化氮（NO₂）是燃烧化石燃料产生的一种空气污染物，也是香烟烟雾的组成成分，被认为与肺气肿等肺部疾病的发病机制有关。为了了解NO₂损害肺部及其功能关键蛋白的机制，以及它与一般蛋白质的反应，我们对α-1-蛋白酶抑制剂（α1PI）、弹性蛋白、聚-L-赖氨酸和聚-L-精氨酸进行了体外暴露实验。当NO₂与α1PI的摩尔比达到400:1及以上时，α1PI抑制其天然生理靶点人中性粒细胞弹性蛋白酶（HNE）的能力下降至对照值的54%。在酶联免疫吸附测定中，当NO₂与α1PI的摩尔比达到100:1及以上时，α1PI与单克隆或多克隆抗体的免疫反应性丧失了50%。对对照和经NO₂处理的α1PI进行的平行邻苯二甲醛和二喹啉甲酸蛋白质测定以及氨基酸分析结果表明，NO₂与赖氨酸残基发生了反应。在生理pH值的水溶液中用NO₂处理之前，先用[³H]HCHO对弹性蛋白和聚-L-赖氨酸的氨基进行还原甲基化标记。弹性蛋白经NO₂处理后，84%的相关放射性物质溶解，经氨基酸分析，其中79%被鉴定为[³H]甲基赖氨酸。聚-L-[³H]赖氨酸经NO₂处理后，凝胶过滤色谱显示50,000 M(r)的聚-L-[³H]赖氨酸已降解为1 - 3000 M(r)的小肽。同样，未标记的聚-L-精氨酸经NO₂处理后，凝胶过滤色谱和总肽分析表明47,500 M(r)的肽也部分降解为小肽。这些结果表明，NO₂与赖氨酸残基的ε-氨基（伯胺）以及肽主链中表面暴露的赖氨酸和精氨酸残基的酰胺氮（仲胺）发生反应，导致肽键断裂。这些发现首次表明了NO₂介导的肽降解，并为体外暴露系统中NO₂损害肺部功能关键蛋白的可能性提供了更多数据。
