Separation, detection, and quantification of hydroperoxides formed at side-chain and backbone sites on amino acids, peptides, and proteins

Research output: Contribution to journalJournal articleResearchpeer-review

Hydroperoxides are major reaction products of radicals and singlet oxygen with amino acids, peptides, and proteins. However, there are few data on the distribution of hydroperoxides in biological samples and their sites of formation on peptides and proteins. In this study we show that normal-or reversed-phase gradient HPLC can be employed to separate hydroperoxides present in complex systems, with detection by postcolumn oxidation of ferrous xylenol orange to the ferric species and optical detection at 560 nm. The limit of detection (10-25 pmol) is comparable to chemiluminescence detection. This method has been used to separate and detect hydroperoxides, generated by hydroxyl radicals and singlet oxygen, on amino acids, peptides, proteins, plasma, and intact and lysed cells. In conjunction with EPR spin trapping and LC/MS/MS, we have obtained data on the sites of hydroperoxide formation. A unique fingerprint of hydroperoxides formed at alpha-carbon (backbone) positions has been identified; such backbone hydroperoxides are formed in significant yields only when the amino acid is part of a peptide or protein. Only side-chain hydroperoxides are detected with free amino acids. These data indicate that free amino acids are poor models of protein damage induced by radicals or other oxidants.

Original languageEnglish
JournalFree Radical Biology & Medicine
Issue number9
Pages (from-to)1279-89
Number of pages11
Publication statusPublished - 1 Nov 2008
Externally publishedYes

    Research areas

  • Amino Acids, Animals, Carbon, Cell Line, Chromatography, High Pressure Liquid, Electron Spin Resonance Spectroscopy, Humans, Hydrogen Peroxide, Hydroxyl Radical, Mass Spectrometry, Mice, Peptides, Proteins

ID: 129670612