Cell and Tissue BiologyCell and Tissue BiologyЦитология0041-37713034-6061The Russian Academy of Sciences66959110.31857/S0041377124030026PERHTFArticlesСтатьиResearch ArticleGenetically Encoded Biosensor HyPer as a Tool for Quantification of Intracellular Hydrogen Peroxide ConcentrationsГенетически кодируемый биосенсор HyPer как инструмент для количественного определения внутриклеточного уровня перекиси водородаLyublinskayaO. G.ЛюблинскаяО. Г.
Russian Federation
o.lyublinskaya@mail.ruIvanovaJ. S.ИвановаЮ. С.
Russian Federation
o.lyublinskaya@mail.ruInstitute of Cytology RASИнститут цитологии РАН1505202466322323327022025Copyright ©; 2024, Russian Academy of SciencesCopyright ©; 2024, Российская академия наук2024Russian Academy of SciencesРоссийская академия наукhttps://journals.eco-vector.com/0041-3771/article/view/669591

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.

В настоящем мини-обзоре систематизирована информация о методах количественной оценки внутриклеточной концентрации перекиси водорода, основанных на использовании генетически кодируемого сенсора пероксида HyPer. Рассматриваются два подхода: 1) градуировка биосенсора с использованием экзогенной перекиси водорода, основанная на оценке скорости проникновения пероксида в клетки и внутриклеточной пероксидазной активности; 2) прямое определение внутриклеточного содержания пероксида, основанное на измерении уровня окисления биосенсора, константы реакции окисления и константы реакции восстановления HyPer в клетках. Применение этих методов позволяет решать широкий спектр задач клеточной редокс-биологии — ранжировать диапазон физиологических и повреждающих концентраций перекиси водорода в клетках, оценивать эффективность системы антиоксидантной защиты в различных клеточных компартментах в условиях окислительного стресса, определять вклад различных ферментативных систем в пероксидазную активность клеток и изучать особенности систем антиоксидантной защиты в различных биологических контекстах (в процессе клеточного старения, дифференцировки, репрограммирования, при развитии патологий). Описанные методы могут быть адаптированы для других генетически-кодируемых биосенсоров перекиси водорода.

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