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。氧化损伤会影响各种氨基酸侧链，在某些情况下，还会导致羰基功能的引入。这些部分特别令人感兴趣，因为它们与2，4-二硝基苯肼反应后用分光光度方法进行定量已成为评估蛋白质氧化的标准方法。本项目的目标之一是研究蛋白质氧化产物的化学特征，特别强调蛋白质的羰基。在以前的进度报告中，我描述了谷氨酸和氨基己二酸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赖氨酸。该方法是基于同位素稀释后的选择离子监测气相色谱/质谱法，并用于蛋白质水解物的分析。分析已经扩展到在体外氧化的其他模型蛋白质(核糖核酸酶和溶菌酶)和新的组织样本。结果证实，在体外金属催化氧化后的蛋白质羰基中，谷氨酸和氨基己二醛占大多数，氧化的牛血清白蛋白仅占总羰基值的一半左右，是一个例外，也不是规则。对大鼠肝脏提取物的分析已经重复，并证实了氨基己二醛和谷氨酸半醛的浓度相似，它们也代表了总羰基值的一半左右。对其他组织蛋白(小鼠的肝、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。重组叙利亚仓鼠SHA(29-231)蛋白的研究始于这样一个事实，即铜(II)结合是该蛋白唯一已知的功能性质，而过渡金属结合促进了MCO。在前一份报告中，我使用标准的抗坏血酸/铜++体系描述了蛋白质对氧化的显著敏感性。随后，我采用氨基酸分析、同步测序和蛋白质消化-液-质联用相结合的方法，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PrP蛋白聚集和沉淀没有得到证实，并确定在所用的实验条件下，仅铜结合就足以引起这种变化。这些结果表明，金属催化的普鲁恩蛋白的氧化很容易发生，这一反应在体内的可能发生和意义应该被考虑。

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.