Characterization of Products of Protein Oxidation

金属催化的蛋白质氧化被认为 在生理病理学和衰老中起重要作用。氧化 损害包括将羰基官能团引入某些氨基 酸性侧链。羰基可以方便地测量 在它们与 二硝基苯肼利用这种方法，我们的实验室 在过去，羰基含量的增加， 随着年龄的增长，细胞内蛋白质，无论是在动物和 人类然而，含羰基的 氧化产物未知。本项目旨在 这类产品的化学特性， 测量的分析技术，以及 这些用于组织和单个蛋白质的分析。从 以前的研究，我们知道精氨酸和脯氨酸被氧化， 谷氨酸和赖氨酸转化为己二酸半醛。目前， 全面介绍了谷氨酸半醛的分析方法， 开发，而己二酸半醛是在先进的 阶段这两种化合物在蛋白质水解产物中通过 选择离子监测气相色谱/质谱 同位素稀释后。首先将样品还原至水解 稳定形式，羟氨基戊酸（HAVA）和 羟基氨基己酸（HACA），然后将其 转化为它们的三氟乙酰基-甲酯挥发性衍生物; 天然丰度和氘代丰度的合成和校准 HAVA的标准已经完成， 谷氨酰胺合成酶在体外氧化和大鼠肝蛋白。 初步结果表明，实际上，HAVA占 在人工氧化的化合物中， 谷氨酰胺氧化酶。定性和定量信息 通过这些研究获得的信息将有助于更好地了解 氧化修饰的性质、对 个体蛋白质，以及其与病理生理学和衰老的关系。

Metal-catalyzed oxidation of proteins is believed to play an important role in physiopathology and aging. Oxidative damage includes introduction of carbonyl functions to certain amino acid side chains. Carbonyls can be conveniently measured spectrophotometrically after their reaction with dinitrophenylhydrazine. Using this method, our laboratory has shown in the past an increase of the carbonyl content of whole intracellular proteins with increased age, both in animals and humans. However, the precise nature of the carbonyl-bearing oxidation products is not known. The present project is aimed at the chemical characterization of such products, the development of analytical techniques for their measurement, and the application of these to the analysis of tissues and individual proteins. From previous research, we know that arginine and proline are oxidized to glutamic and lysine to adipic semialdehydes. At the present time, an analytical method for glutamic semialdehyde has been fully developed, while one for adipic semialdehyde is at an advanced stage. Both compounds are measured in protein hydrolysates by selected ion monitoring gas chromatography/mass spectrometry after isotopic dilution. Samples are first reduced to the hydrolysis stable forms, hydroxyaminovaleric acid (HAVA) and hydroxyaminocaproic acid (HACA), respectively, which are then converted to their trifluoroacetyl-methyl ester volatile derivatives; synthesis and calibration of natural abundance and deuterated standards has been completed for HAVA, which has been measured in glutamine synthetase oxidized in vitro and in rat liver proteins. Preliminary results indicate that, in fact, HAVA accounts for a substantial proportion of carbonyl groups in artificially oxidized glutamine oxidase. The qualitative and quantitative information gained through these studies will be useful to better understand the nature of the oxidative modifications, the extent of damage to individual proteins, and its bearing to physiopathology and aging.