QUANTITATION OF OXIDATION-INDUCED CONFORMATIONAL CHANGES BY 1ST ORDER REACTIONS

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。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 用于羟基自由基足迹法的最流行和良好描述的技术使用用于产生羟基自由基的技术，所述羟基自由基导致大于1毫秒的自由基寿命，这对于蛋白质经历剧烈的氧化诱导的构象变化和待探测的非天然结构来说是足够的时间。 大多数在大于一毫秒的时间尺度上产生羟基自由基的技术发生在伪一级条件下。 通常，该反应被严格限制为仅允许发生非常少量的氧化（~ 1氧化/分子），或者氧化诱导的变化之后是诸如光谱学的方法，其需要比羟基自由基足迹法多得多的样品。 我们正在开发一个 使用完整蛋白质氧化产物的分析来检测和定量在一级羟基自由基足迹实验期间发生的氧化诱导的解折叠的量的方法。

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. The most popular and well-described techniques for hydroxyl radical footprinting use techniques for generating hydroxyl radicals that result in a radical lifetime of greater than one millisecond, which is sufficient time for a protein to experience a drastic oxidation-induced conformational change and the non-native structure to be probed. Most techniques generating hydroxyl radicals on a timescale greater than one millisecond occur under pseudo-first order conditions. Typically, the reaction is either strictly limited to only allow a very small amount of oxidation to occur (~ 1 oxidation/molecule), or oxidation-induced changes are followed by methods such as optical spectroscopy, which require substantially more sample than hydroxyl radical footprinting. We are developing a method for detecting and quantitating the amount of oxidation-induced unfolding that occurs during a first order hydroxyl radical footprinting experiment using analysis of the intact protein oxidation products.