Genetically Encoded Biosensor HyPer as a Tool for Quantification of Intracellular Hydrogen Peroxide Concentrations

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Abstract

This mini-review systematizes information on methods for quantitative assessment of intracellular hydrogen peroxide concentration based on the use of a genetically encoded peroxide sensor HyPer. Two approaches are being considered: 1) calibration of the biosensor using exogenous hydrogen peroxide, based on assessing the rate of peroxide penetration into cells and intracellular peroxidase activity; 2) direct determination of the intracellular peroxide content, based on measuring the level of oxidation of the biosensor, the oxidation reaction constant and the reduction reaction constant of HyPer in the cells. The use of these methods makes it possible to solve a wide range of tasks in cellular redox biology — to determine the range of physiological and damaging concentrations of hydrogen peroxide in cells, to evaluate the effectiveness of the antioxidant defense system in various cellular compartments under conditions of oxidative stress, to determine the contribution of various enzymatic systems to the peroxidase activity of cells, and to characterize antioxidant defense systems in various biological contexts (in the process of cellular senescence, differentiation, reprogramming, during the development of pathologies). The described methods can be adapted for other genetically encoded hydrogen peroxide biosensors.

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About the authors

O. G. Lyublinskaya

Institute of Cytology RAS

Author for correspondence.
Email: o.lyublinskaya@mail.ru
Russian Federation, St. Petersburg, 194064, Tikhoretskii pr., 4

J. S. Ivanova

Institute of Cytology RAS

Email: o.lyublinskaya@mail.ru
Russian Federation, St. Petersburg, 194064, Tikhoretskii pr., 4

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2. Fig. 1. Scheme of experiment on HyPer biosensor calibration in living cells using exogenous H2O2. Designations: gradient is the ratio of extracellular concentration of H2O2 ([H2O2]ex) to intracellular concentration ([H2O2]in); kPXi is the rate constant of elimination of H2O2 by intracellular peroxidases: peroxyredoxins (Prx), glutathione peroxidases (GPx), catalase (Cat), c1; kperm is the rate constant of penetration of H2O2 into the cell, c1; HyPer ratio is the ratiometric biosensor signal, the ratio of HyPer fluorescence signals at two different excitation wavelengths: 488 nm (I488) and 405 nm (I405).

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3. 2. Experimental scheme for determining the intracellular concentration of H2O2 ([H2O2]in) during the induction of external oxidative stress. The left panel shows the measurement scheme, flow cytometric analysis of cells. The right panel shows the processing of experimental data, an example of a kinetic curve (black) and the approximation of this curve by kinetic equation (4) (red curve). Designations: I488 and I405 are HyPer fluorescence signals at excitation wavelengths of 488 and 405 nm, respectively; OxD (oxidation degree) is the ratio of the number of oxidized HyPer molecules to their total number in the cell; kox is the rate constant of the second—order reaction between HyPer and H2O2, c1.

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