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

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.

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: HyPer_rd reduced form of HyPer; HyPer_ox, 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

Scheme describing the method for quantification of exogenous and endogenous H₂O₂ concentrations in HyPer-expressing cells. Abbreviations: HyPer_ox and HyPer_rd, oxidized and reduced HyPer fractions; k_ox and k_rd, 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

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

Kinetics of HyPer oxidation in K562 cells exposed to H₂O₂. (A) Reduced HyPer fraction drop with time after H₂O₂ addition to cell suspension. HyPer_rd 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: HyPer_rd, reduced HyPer fraction calculated with Eq. (9); k_ox and k_rd, apparent first-order rate constants of HyPer oxidation and reduction, respectively; [H₂O₂]_ex, extracellular peroxide concentration.