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, 39 (1), 26-30

Kinetic Approaches to Measuring Peroxiredoxin Reactivity


Kinetic Approaches to Measuring Peroxiredoxin Reactivity

Christine C Winterbourn et al. Mol Cells.


Peroxiredoxins are ubiquitous thiol proteins that catalyse the breakdown of peroxides and regulate redox activity in the cell. Kinetic analysis of their reactions is required in order to identify substrate preferences, to understand how molecular structure affects activity and to establish their physiological functions. Various approaches can be taken, including the measurement of rates of individual steps in the reaction pathway by stopped flow or competitive kinetics, classical enzymatic analysis and measurement of peroxidase activity. Each methodology has its strengths and they can often give complementary information. However, it is important to understand the experimental conditions of the assay so as to interpret correctly what parameter is being measured. This brief review discusses different kinetic approaches and the information that can be obtained from them.

Keywords: hyperoxidation; kinetics; peroxide; peroxiredoxin; thiol oxidation.


Fig. 1.
Fig. 1.
Possible redox-regulatory mechanisms for peroxiredoxins. T, oxidant-sensitive target.
Fig. 2.
Fig. 2.
Redox transformations of a typical 2Cys Prx. Sp and Sr refer to the peroxidatic and resolving Cys respectively; Trx, thioredoxin; TrxR, thioredoxin reductase. Oxidation at two active sites of the homodimer is shown. Although interaction between the two is possible, in this article we have considered the kinetics of two acting independently. Numbers in parenthesis refer to the reactions shown and are described in the text.
Fig. 3.
Fig. 3.
Competitive kinetics with HRP for measuring Prx rate constants. (A) Principle; (B) Example showing spectral changes and calculation of the rate constant for Prx3. (C) Inhibition of Prx2 oxidation by increasing concentrations of catalase as shown by non-reducing SDS-PAGE. Data are from (Peskin et al., 2007) and (Cox et al., 2009b).

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