Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics


Autoria(s): TOLEDO JR., Jose Carlos; AUDI, Renata; OGUSUCU, Renata; MONTEIRO, Gisele; SOARES NETTO, Luis Eduardo; AUGUSTO, Ohara
Contribuinte(s)

UNIVERSIDADE DE SÃO PAULO

Data(s)

20/10/2012

20/10/2012

2011

Resumo

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) x 10(7) M(-1) s(-1)) and peroxynitrite ((3.7 +/- 0.4) x 10(5) M(-1) s(-1)) at pH 7.4 and 25 degrees C. (C) 2011 Elsevier Inc. All rights reserved.

Fundacao de Amparo A Pesquisa do Estado de Sao Paulo (FAPESP)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Identificador

FREE RADICAL BIOLOGY AND MEDICINE, v.50, n.9, p.1032-1038, 2011

0891-5849

http://producao.usp.br/handle/BDPI/30925

10.1016/j.freeradbiomed.2011.02.020

http://dx.doi.org/10.1016/j.freeradbiomed.2011.02.020

Idioma(s)

eng

Publicador

ELSEVIER SCIENCE INC

Relação

Free Radical Biology and Medicine

Direitos

restrictedAccess

Copyright ELSEVIER SCIENCE INC

Palavras-Chave #Reactive cysteines #Hydrogen peroxide #Hydrogen peroxide sensors #Horseradish peroxidase #Peroxiredoxin kinetics #rPrdx6 kinetics #Free radicals #SENSITIVE THIOL PROTEINS #SULFENIC ACID FORMATION #HYDROGEN-PEROXIDE #OXIDATIVE STRESS #DEPENDENT PEROXIDASE #PEROXIREDOXINS #CELL #PEROXYNITRITE #H2O2 #MECHANISMS
Tipo

article

original article

publishedVersion