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Using the heme peroxidase APEX2 to probe intracellular H₂O₂ flux and diffusion.

ABSTRACT: Currently available genetically encoded H₂O₂ probes report on the thiol redox state of the probe, which means that they reflect the balance between probe thiol oxidation and reduction. Here we introduce the use of the engineered heme peroxidase APEX2 as a thiol-independent chemogenetic H₂O₂ probe that directly and irreversibly converts H₂O₂ molecules into either fluorescent or luminescent signals. We demonstrate sensitivity, specificity, and the ability to quantitate endogenous H₂O₂ turnover. We show how the probe can be used to detect changes in endogenous H₂O₂ generation and to assess the roles and relative contributions of endogenous H₂O₂ scavengers. Furthermore, APEX2 can be used to study H₂O₂ diffusion inside the cytosol. Finally, APEX2 reveals the impact of commonly used alkylating agents and cell lysis protocols on cellular H₂O₂ generation.

SUBMITTER: Eid M

PROVIDER: S-EPMC10858230 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Using the heme peroxidase APEX2 to probe intracellular H2O2 flux and diffusion.

Eid Mohammad M Barayeu Uladzimir U Sulková Kateřina K Aranda-Vallejo Carla C Dick Tobias P TP

Nature communications 20240209 1

Currently available genetically encoded H2O2 probes report on the thiol redox state of the probe, which means that they reflect the balance between probe thiol oxidation and reduction. Here we introduce the use of the engineered heme peroxidase APEX2 as a thiol-independent chemogenetic H2O2 probe that directly and irreversibly converts H2O2 molecules into either fluorescent or luminescent signals. We demonstrate sensitivity, speci ...[more]

PMID: 38336829

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