PROTEIN HYDROPEROXIDES AS SECONDARY OXIDANTS IN HYDROXYL RADICAL FOOTPRINTING

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 以前的研究表明，含亮氨酸的肽氧化形成的肽基氢过氧化物的亮氨酸侧链。 肽基氢过氧化物以前从未在羟基自由基足迹实验中观察到。 以前认为它们太不稳定而不能在蛋白质制备和质谱分析中存活;然而，通过质谱在含亮氨酸的肽中发现它们表明它们足够稳定以在溶液中持续存在。 如果它们确实存在于溶液中，它们可能能够进行含硫侧链的直接双电子氧化，导致羟基自由基足迹实验中的假阳性。 此外，用于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法在去除肽基氢过氧化物方面是无效的。 我们设计并进行了一系列实验，以确定肽基氢过氧化物是否是各种氨基酸氧化的常见产物，以确定这些肽基氢过氧化物是否能够对甲硫氨酸残基进行双电子氧化，并确定过氧化氢酶处理或用甲硫氨酸酰胺清除是否有效消除肽基氢过氧化物。

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.