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. 2019 Jun:24:101200.
doi: 10.1016/j.redox.2019.101200. Epub 2019 Apr 16.

Measuring intracellular concentration of hydrogen peroxide with the use of genetically encoded H2O2 biosensor HyPer

Olga Lyublinskaya  1 , Fernando Antunes  2
Affiliations

Measuring intracellular concentration of hydrogen peroxide with the use of genetically encoded H2O2 biosensor HyPer

Olga Lyublinskaya et al. Redox Biol. 2019 Jun.

Abstract

In this study, we propose a method for quantification of average hydrogen peroxide concentration within a living cell that is based on the use of genetically encoded H2O2 biosensor HyPer. The method utilizes flow cytometric measurements of HyPer fluorescence in H2O2-exposed cells to analyze the biosensor oxidation kinetics. Fitting the experimental curves with kinetic equations allows determining the rate constants of HyPer oxidation/reduction which are used further for the calculation of peroxide concentrations in the cells of interest both in the presence and absence of external H2O2. Applying this method to K562 cells, we have estimated the gradient as about 390-fold between the extracellular and intracellular level of exogenous H2O2 in cells exposed to the micromole doses of peroxide, as well as the average basal level of H2O2 in the cytosol of undisturbed cells ( [Formula: see text] ). The method can be extended to other H2O2-sensitive redox probes or to procedures in which, rather than adding external peroxide, intracellular production of peroxide is triggered, providing a tool to quantitate not only basal average H2O2 concentrations but also the concentration of peroxide build up in the vicinity of redox probes.

Keywords: Genetically encoded biosensors; Gradient; H(2)O(2); HyPer; Hydrogen peroxide; Kinetics; Rate constants.

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Figures

Fig. 1
Fig. 1
Analysis of HyPer fluorescence in K562 cells exposed to extracellular H2O2. (A) Scheme demonstrating the changes in the excitation spectrum of HyPer upon oxidation. (B) Flow cytometry histograms of K562 cells measured after two-minutes exposure to different concentrations of H2O2. (C) Dependence of the mean EX488/FL525 HyPer signal intensity on the extracellular H2O2 concentration applied to cells. Abbreviations: HyPerrd reduced form of HyPer; HyPerox, oxidized form of HyPer; HyPer FL, HyPer fluorescence; EX488/FL525, HyPer green fluorescence signal measured at blue laser (488 nm) excitation; [H2O2]ex, extracellular peroxide concentration.
Fig. 2
Fig. 2
Scheme describing the method for quantification of exogenous and endogenous H2O2 concentrations in HyPer-expressing cells. Abbreviations: HyPerox and HyPerrd, oxidized and reduced HyPer fractions; kox and krd, apparent first-order rate constants of HyPer oxidation and reduction; [H2O2]basal basal endogenous hydrogen peroxide concentration, [H2O2]addin, additional intracellular hydrogen peroxide concentration arising from the cell exposure to external peroxide.
Fig. 3
Fig. 3
Quantitation of the reduced HyPer fraction in cells using total reduction/oxidation of the sensor. (A, B) Flow cytometry histograms of HyPer-expressing (A) and SypHer-expressing (B) K562 cells: untreated and exposed to high doses of DTT (10 mM, 10min) or H2O2 (0.5 mM, 5min).
Fig. 4
Fig. 4
Kinetics of HyPer oxidation in K562 cells exposed to H2O2. (A) Reduced HyPer fraction drop with time after H2O2 addition to cell suspension. HyPerrd was estimated using Eq. (9). Data fitting with Eq. (1) is shown in red. (B, C) Dependence of the apparent first-order rate constants of HyPer oxidation (B) and reduction (C) on extracellular H2O2 concentration. Both dependencies were derived from fittings of kinetic curves (C). Abbreviations: HyPerrd, reduced HyPer fraction calculated with Eq. (9); kox and krd, apparent first-order rate constants of HyPer oxidation and reduction, respectively; [H2O2]ex, extracellular peroxide concentration.
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