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。氧化损伤影响多种氨基酸侧链，在某些情况下，导致羰基功能的引入。与2,4-二硝基苯肼反应后，用分光光度法定量测定这些部分已成为评估蛋白质氧化的标准方法。本项目的一个目标是蛋白质氧化产物的化学表征，特别强调蛋白质羰基。在之前的进展报告中，我描述了用于定量谷氨酸和氨基己二半醛的分析方法的发展，这两种特定的羰基产物分别来自精氨酸和脯氨酸以及赖氨酸。该方法基于同位素稀释后选择的离子监测气相色谱/质谱法，并应用于蛋白质水解物。分析已经扩展到额外的模型蛋白（rna酶和溶菌酶）在体外氧化和新的组织样品。结果证实，在体外金属催化氧化后，谷氨酸和氨基己二半醛占蛋白质羰基的大部分，而氧化的牛血清白蛋白只占总羰基值的一半左右，这是一个例外，而不是规则。对大鼠肝脏提取物的重复分析证实，氨基己二醛和谷氨酸半醛以相似的浓度存在，它们也占总羰基值的一半左右。对其他组织蛋白（小鼠肝、肺和脑）的分析正在进行中。该项目的第二个目标是表征特定蛋白质的氧化修饰和损伤。考虑到铜（II）结合是重组叙利亚仓鼠SHa（29-231）朊病毒蛋白唯一已知的功能特性，并且过渡金属结合有助于MCO，我们开始了对该朊病毒蛋白的研究。在之前的报告中，我描述了使用标准抗坏血酸/Cu++体系的蛋白质对氧化的惊人敏感性。随后，我使用氨基酸分析、同步测序和蛋白水解消化结合液相色谱/质谱法研究了蛋白质不同结构域对氧化的敏感性。初步结果表明，氧化导致朊病毒蛋白聚集和沉淀尚未得到证实，并且确定在所用的实验条件下，铜的单独结合足以诱导这种变化。这些结果表明，金属催化的朊蛋白氧化很容易发生，应该考虑这种反应在体内可能发生的情况及其影响。

Metal-catalyzed oxidation (MCO) of proteins is believed to play an important role in physiopathology and aging. Oxidative damage affects a variety of amino acid side chains and, in some instances, results in introduction of carbonyl functions. These moieties are of particular interest as their quantification by spectrophotometric methods after reaction with 2,4-dinitrophenylhydrazine has become the standard method to assess protein oxidation. One objective of the present project is the chemical characterization of protein oxidation products, with special emphasis on protein carbonyls. In previous progress reports, I described the development of analytical methods for quantitation of glutamic and aminoadipic semialdehydes, two specific carbonyl products derived from arginine and proline an from lysine, respectively. The methods are based on selected ion monitoring gas chromatography/mass spectrometry after isotopic dilution and are applied to protein hydrolysates. Analyses have been extended to additional model proteins (RNase and lysozyme) oxidized in vitro and to new tissue samples. Results confirm that glutamic and aminoadipic semialdehydes account for the majority of protein carbonyls after metal-catalyzed oxidation in vitro and that oxidized bovine serum albumin, in which they make up only about one-half of the total carbonyl value, is an exception and not the rule. Analyses of rat liver extracts have been repeated and have confirmed that aminoadipic and glutamic semialdehydes are present at similar concentrations, and that they represent also about one-half of the total carbonyl value. Analyses of additional tissue proteins (mouse liver, lung, and brain) are in progress. The second objective of this project is the characterization of oxidative modification and damage in specific proteins. Studies on recombinant Syrian hamster SHa(29-231) prion protein were initiated given the fact that copper (II) binding is the only known functional property of the prion protein, and that transition metal binding facilitates MCO. In the previous report, I described the striking susceptibility of the protein to oxidation using the standard ascorbate/Cu++ system. Subsequently, I have studied the susceptibility to oxidation of different domains of the protein using a combination of amino acid analysis, simultaneous sequencing, and proteolytic digestion coupled to liquid chromatography/mass spectrometry. Preliminary results indicating that oxidation leads to prion protein aggregation and precipitation were not confirmed, and it was determined that copper binding alone is sufficient to induce such changes under the experimental conditions used. These results indicate that metal-catalyzed oxidation of the prion protein occurs easily, and that the possible occurrence and implications of this reaction in vivo should be considered.