PROTEIN HYDROPEROXIDES AS SECONDARY OXIDANTS IN HYDROXYL RADICAL FOOTPRINTING

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 先前对含亮氨酸多肽的氧化研究表明，亮氨酸侧链上会形成一种多肽过氧化氢。在羟基足迹实验中，从来没有观察到过肽基过氧化氢。以前人们认为它们太不稳定，不能在蛋白质制备和质谱分析中存活；然而，通过质谱学在含有亮氨酸的多肽中发现它们，表明它们足够稳定，可以在溶液中坚持。如果它们确实存在于溶液中，它们可能能够对含硫侧链进行直接两电子氧化，从而导致羟基足迹实验中的假阳性。此外，用来去除过氧化氢的标准方法，包括过氧化氢酶处理和液相色谱分析，在去除肽基过氧化氢方面将无效。我们设计并进行了一系列实验，以确定肽基过氧化氢是否是各种氨基酸氧化的常见产物，确定这些肽基过氧化氢是否具有双电子氧化蛋氨酸残基的能力，以及确定过氧化氢酶处理或蛋氨酸酰胺清除是否有效地清除肽基过氧化氢。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Previous studies of oxidation of leucine-containing peptides revealed the formation of a peptidyl hydroperoxide on the leucine side chain. Peptidyl hydroperoxides have never before been observed in hydroxyl radical footprinting experiments. It was previously thought that they were too unstable to survive the protein preparation and mass spectrometry analysis; however, their discovery in the leucine-containing peptide by mass spectrometry indicates that they are sufficiently stable to persist in solution. If they do exist in solution, they may be capable of performing direct two-electron oxidation of sulfur-containing side chains, causing false positives in hydroxyl radical footprinting experiments. Additionally, standard methods used to remove hydrogen peroxide, including catalase treatment and liquid chromatography would be ineffective at removing peptidyl hydroperoxides. We designed and carried out a series of experiments to determine if peptidyl hydroperoxides are common products of oxidation of various amino acids, to determine if these peptidyl hydroperoxides are capable of two-electron oxidation of methionine residues, and to determine if catalase treatment or scavenging with methionine amide is effective at eliminating the peptidyl hydroperoxides.