Aliphatic peptidyl hydroperoxides as a source of secondary oxidation in hydroxyl radical protein footprinting |
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Authors: | Jessica Saladino Mian Liu David Live Joshua S Sharp |
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Institution: | 1. Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA
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Abstract: | Hydroxyl radical footprinting is a technique for studying protein structure and binding that entails oxidizing a protein system
of interest with diffusing hydroxyl radicals, and then measuring the amount of oxidation of each amino acid. One important
issue in hydroxyl radical footprinting is limiting amino acid oxidation by secondary oxidants to prevent uncontrolled oxidation,
which can cause amino acids to appear more solvent accessible than they really are. Previous work suggested that hydrogen
peroxide was the major secondary oxidant of concern in hydroxyl radical footprinting experiments; however, even after elimination
of all hydrogen peroxide, some secondary oxidation was still detected. Evidence is presented for the formation of peptidyl
hydroperoxides as the most abundant product upon oxidation of aliphatic amino acids. Both reverse phase liquid chromatography
and catalase treatment were shown to be ineffective at eliminating peptidyl hydroperoxides. The ability of these peptidyl
hydroperoxides to directly oxidize methionine is demonstrated, suggesting the value of methionine amide as an in situ protectant.
Hydroxyl radical footprinting protocols require the use of an organic sulfide or similar peroxide scavenger in addition to
removal of hydrogen peroxide to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation
of sulfur-containing residues. |
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