Trujillo Madia, Ferrer-Sueta Gerardo, Radi Rafael
Department of Biochemistry, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
Methods Enzymol. 2008;441:173-96. doi: 10.1016/S0076-6879(08)01210-X.
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.
过氧化物酶催化将过氧亚硝酸盐还原为亚硝酸盐。过氧化物酶的过氧化物酶活性半胱氨酸与过氧亚硝酸盐迅速反应。该反应的速率常数可以使用停流分光光度计通过初始速率法或稳态测量直接跟踪300至310nm区域内过氧亚硝酸盐的消失来测量，或者通过与已知速率常数的反应竞争来测量。用于与过氧化物酶竞争的反应包括过氧亚硝酸盐对锰（III）卟啉和辣根过氧化物酶的氧化。此外，还描述了一种方法，其中过氧化氢与过氧亚硝酸盐竞争过氧化物酶的氧化。此外，还描述了一种用于确定硫氧还蛋白介导的过氧化物酶还原动力学以完成催化循环的荧光技术。所综述的所有方法都提供了可靠的速率常数，并且可以结合使用它们来提供进一步的保证；讨论了不同方法的适用性和优点。
