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

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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.