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Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics
- Source :
-
Free Radical Biology & Medicine . May2011, Vol. 50 Issue 9, p1032-1038. 7p. - Publication Year :
- 2011
-
Abstract
- 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)×107 M−1 s−1) and peroxynitrite ((3.7±0.4)×105 M−1 s−1) at pH 7.4 and 25°C. [Copyright &y& Elsevier]
Details
- Language :
- English
- ISSN :
- 08915849
- Volume :
- 50
- Issue :
- 9
- Database :
- Academic Search Index
- Journal :
- Free Radical Biology & Medicine
- Publication Type :
- Academic Journal
- Accession number :
- 59774084
- Full Text :
- https://doi.org/10.1016/j.freeradbiomed.2011.02.020