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Development of analysis for all biologically relevant derivatives of NO

开发所有生物学相关的 NO 衍生物的分析

基本信息

批准号：
12557010
负责人：
AKAIKE Takaaki
金额：
$ 3.01万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

Reactive nitrogen oxides derived from NO produce nitration adducts of various biological molecules. We have developed a series of methods for identification and quantification of nitrated adducts of amino acids and nucleic acid bases, such as 3-nitro-L-tyrosine, nitro-L-tryptophan, and 8-nitroguanine. The assay couples an HPLC system with electrochemical (EC) analysis. Each nitrated compound in Pronase-digested samples, being separated by reverse-phase HPLC, was first reduced electrochemically at -900 mV, followed by detection with an analytical cell at +300 mV. Detection limit for 3-nitrotyrosine reached 10^<-15>mol. Increased protein-bound 3-nitrotyrosine was demonstrated in bronchoalveolar lavage from mice infected with influenza virus; whereas protein-bound 3-nitrotyrosine was not identified with mice deficient in inducible NO synthase. Similarly, appreciable amount of 3-nitrotyrosine was detected in sputum from patients with bronchial asthma and chronic bronchitis. The level of nitrotryptophan was, however, below detection limit in both murine and human samples. Also, our immunohistochemical analysis with a specific anti-3-nitroguanine antibody clearly showed formation of 3-nitroguanosine in the bronchial epithelial cells of the virus-infected mouse lungs. This is the first demonstration of a nitrated nucleotide base formed endogenously in biological systems. The present methods may help us to better understand the biological relevance and the mechanism of biological nitration.

由NO衍生的活性氮氧化物产生各种生物分子的硝化加合物。我们已经发展了一系列鉴定和定量氨基酸和核酸碱基的硝化加合物的方法，如3-硝基-L-酪氨酸、硝基-L-色氨酸和8-硝基鸟嘌呤。该测定将HPLC系统与电化学（EC）分析耦合。链霉蛋白酶消化样品中的每种硝化化合物，通过反相HPLC分离，首先在-900 mV下电化学还原，然后在+300 mV下用分析电池检测。3-硝基酪氨酸的检测限可达10 μ <-15>mol.在流感病毒感染小鼠的支气管肺泡灌洗液中证实了蛋白结合3-硝基酪氨酸的增加;而在诱导型NO合酶缺乏的小鼠中未发现蛋白结合3-硝基酪氨酸。同样，在支气管哮喘和慢性支气管炎患者的痰中检测到相当数量的3-硝基酪氨酸。然而，在鼠和人样品中，硝基色氨酸的水平低于检测限。此外，我们的免疫组化分析与一个特定的抗3-硝基鸟嘌呤抗体清楚地表明，形成3-硝基鸟苷在支气管上皮细胞的病毒感染的小鼠肺。这是第一次证明硝化核苷酸碱基在生物系统中内源性形成。这些方法可以帮助我们更好地理解生物硝化的生物相关性和机理。