CHARACTERIZATION OF PRODUCTS OF PROTEIN OXIDATION

蛋白质的金属催化氧化（MCO）被认为在生理病理和衰老中起着重要作用。氧化损伤包括在某些氨基酸侧链上引入羰基功能。本项目旨在研究蛋白质氧化产物的化学特性，特别强调蛋白质羰基。自上期进度报告以来，完成了己二半醛的定量分析方法。因此，在同位素稀释后，通过选择离子监测气相色谱/质谱法，现在可以在还原蛋白水解物中常规测量谷氨酸和己二半醛。对体外氧化的两种模型蛋白谷氨酰胺合成酶（GS）和牛血清白蛋白（BSA）的分析表明，这两种半醛合在一起约占氧化GS中蛋白质羰基的总数，而它们占氧化BSA中蛋白质羰基的约60%。新方法与经典的蛋白质羰基分析相结合，在研究中也被证明是有用的，这些研究表明共价修饰增强了蛋白质的金属结合能力，使它们更容易受到MCO的影响。因此，蛋白质的赖氨酸残基转化为羧甲基赖氨酸衍生物，如发生在糖尿病患者和老年人中，导致mco介导的羰基和己二/谷氨酸半醛衍生物的形成增加。在先前报告中对大鼠肝组织的研究的基础上，我们还在健康供体和早衰症患者培养的人类成纤维细胞中测量了谷氨酸和己二醛半醛。同时，对不同年龄果蝇的线粒体蛋白提取物进行了分析。这些研究提供了特定羰基产物浓度与衰老之间的相关性信息。一个重要的结果是，与体外氧化的蛋白质相比，谷氨酸和己二半醛对蛋白质羰基的贡献要小得多。研究单个蛋白质的氧化也是我们的目的。在这方面，已经开始了重组仓鼠朊病毒蛋白氧化的研究。初步结果表明，MCO诱导了这种聚集和沉淀的蛋白质构象的剧烈变化。-金属催化氧化，蛋白质羰基，老化，谷氨酸半醛，己二半醛，羧甲基赖氨酸。

Metal-catalyzed oxidation (MCO) 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. The present project is aimed at the chemical characterization of protein oxidation products, with special emphasis on protein carbonyls. Since the last progress report, the analytical method for quantitation of adipic semialdehyde was completed. Thus, both glutamic and adipic semialdehydes can now be routinely measured in reduced protein hydrolysates by selected ion monitoring gas chromatography/mass spectrometry after isotopic dilution. Analysis of two model proteins, glutamine synthetase (GS) and bovine serum albumin (BSA), oxidized in vitro indicated that the combined two mentioned semialdehydes make up approximately the totality of protein carbonyls in oxidized GS, while they account for ~60% of protein carbonyls in oxidized BSA. The new procedures, in combination with the classic protein carbonyl assay, proved useful also in studies showing that covalent modifications that enhance the metal binding capacity of proteins make them more susceptible to MCO. Thus, conversion of lysine residues of proteins to carboxymethyl-lysine derivatives, as occurs in patients with diabetes and in aging, led to an increase in MCO-mediated formation of carbonyl groups and adipic/glutamic semialdehyde derivatives. Expanding the studies of rat liver tissue described in the previous report, glutamic and adipic semialdehydes were also measured in human cultured fibroblasts from healthy donors and patients affected by progeria. Also, mitochondrial protein extracts from flies of different ages were analyzed. These studies are providing information on the correlation between concentrations of specific carbonyl products and aging. One important result emerging is that, in contrast to proteins oxidized in vitro, glutamic and adipic semialdehydes represent a much more modest contribution to the total of protein carbonyls. It is also our purpose to study the oxidation of individual proteins. In this respect, studies on the oxidation of recombinant hamster prion protein have been initiated. Preliminary results indicate that MCO induces a dramatic change in the conformation of this protein that aggregates and precipitates. - Metal-catalyzed oxidation, protein carbonyls, aging, glutamic semialdehyde, adipic semialdehyde, carboxymethyl-lysine.