title: Real-time measurements of amino acid and protein hydroperoxides using coumarin boronic acid.
abstract: Hydroperoxides of amino acid and amino acid residues (tyrosine, cysteine, tryptophan, and histidine) in proteins are formed during oxidative modification induced by reactive oxygen species. Amino acid hydroperoxides are unstable intermediates that can further propagate oxidative damage in proteins. The existing assays (oxidation of ferrous cation and iodometric assays) cannot be used in real-time measurements. In this study, we show that the profluorescent coumarin boronic acid (CBA) probe reacts with amino acid and protein hydroperoxides to form the corresponding fluorescent product, 7-hydroxycoumarin. 7-Hydroxycoumarin formation was catalase-independent. Based on this observation, we have developed a fluorometric, real-time assay that is adapted to a multiwell plate format. This is the first report showing real-time monitoring of amino acid and protein hydroperoxides using the CBA-based assay. This approach was used to detect protein hydroperoxides in cell lysates obtained from macrophages exposed to visible light and photosensitizer (rose bengal). We also measured the rate constants for the reaction between amino acid hydroperoxides (tyrosyl, tryptophan, and histidine hydroperoxides) and CBA, and these values (7-23 M(-1) s(-1)) were significantly higher than that measured for H2O2 (1.5 M(-1) s(-1)). Using the CBA-based competition kinetics approach, the rate constants for amino acid hydroperoxides with ebselen, a glutathione peroxidase mimic, were also determined, and the values were within the range of 1.1-1.5 × 10(3) M(-1) s(-1). Both ebselen and boronates may be used as small molecule scavengers of amino acid and protein hydroperoxides. Here we also show formation of tryptophan hydroperoxide from tryptophan exposed to co-generated fluxes of nitric oxide and superoxide. This observation reveals a new mechanism for amino acid and protein hydroperoxide formation in biological systems.
authors.affiliation: From the Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and the Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors.forename: Radoslaw
authors.initials: R
authors.lastname: Michalski
authors.affiliation: From the Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and the Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors.forename: Jacek
authors.initials: J
authors.lastname: Zielonka
authors.affiliation: the Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Gapys
authors.affiliation: the Institute of Applied Radiation Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Marcinek
authors.affiliation: From the Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and.
authors.forename: Joy
authors.initials: J
authors.lastname: Joseph
authors.affiliation: From the Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and balarama@mcw.edu.
authors.forename: Balaraman
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry; D001393:Azoles; D001897:Boronic Acids; D003199:Computer Systems; D003374:Coumarins; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D054833:Isoindoles; D016566:Organoselenium Compounds; D010545:Peroxides / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry
chemical_list: D000596:Amino Acids; D001393:Azoles; D001897:Boronic Acids; D003374:Coumarins; D005456:Fluorescent Dyes; D054833:Isoindoles; D016566:Organoselenium Compounds; D010545:Peroxides; D011506:Proteins; C042986:ebselen
keywords: Amino Acid Hydroperoxides; Boronates; Fluorescence; Free Radicals; Hydroperoxides; Oxidative Stress; Oxygen Radicals; Protein Chemical Modification; Singlet Oxygen; Tyrosine Hydroperoxide