title: Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics.
abstract: Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 ± 0.2) × 10⁷M⁻¹ s⁻¹) and peroxynitrite ((3.7 ± 0.4) × 10⁵ M⁻¹ s⁻¹) at pH 7.4 and 25°C.
authors.affiliation: Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, SP, Brazil. toledo@ffclrp.usp.br
authors.forename: José Carlos
authors.initials: JC
authors.lastname: Toledo
authors.forename: Renata
authors.initials: R
authors.lastname: Audi
authors.forename: Renata
authors.initials: R
authors.lastname: Ogusucu
authors.forename: Gisele
authors.initials: G
authors.lastname: Monteiro
authors.forename: Luis Eduardo Soares
authors.initials: LE
authors.lastname: Netto
authors.forename: Ohara
authors.initials: O
authors.lastname: Augusto
mesh_terms: D000818:Animals; D056246:Chemistry, Analytic; D003001:Cloning, Molecular; D003545:Cysteine / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D020563:Gammaproteobacteria; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D054464:Peroxiredoxins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013053:Spectrophotometry
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; C098326:oxyferryl porphyrin pi cation radical; D030421:Peroxynitrous Acid; D006639:Histidine; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D054464:Peroxiredoxins; D003545:Cysteine; D010100:Oxygen