Kinetic studies on peroxynitrite reduction by peroxiredoxins.
Peroxiredoxins catalytically reduce peroxynitrite to nitrite. The peroxidatic cysteine of peroxiredoxins reacts rapidly with peroxynitrite. The rate constant of that reaction can be measured using a stopped flow spectrophotometer either directly by following peroxynitrite disappearance in the region of 300 to 310 nm using an initial rate approach or steady-state measurements or by competition with a reaction of known rate constant. The reactions used to compete with peroxiredoxins include the oxidation of Mn(III)porphyrins and horseradish peroxidase by peroxynitrite. Additionally, a method is described in which a hydroperoxide competes with peroxynitrite for the oxidation of peroxiredoxin. Moreover, a fluorescent technique for determining the kinetics of thioredoxin-mediated peroxiredoxin reduction, closing the catalytic cycle, is also described. All methods reviewed provide reliable values of rate constants and a combination of them can be used to provide further reassurance; applicability and advantages of the different methodologies are discussed.
Department of Biochemistry, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
Madia
M
Trujillo
Gerardo
G
Ferrer-Sueta
Rafael
R
Radi
D000818:Animals; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D010084:Oxidation-Reduction; D054464:Peroxiredoxins / Q000737:chemistry / Q000493:pharmacokinetics / Q000502:physiology*; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics
D030421:Peroxynitrous Acid; D054464:Peroxiredoxins