Dataset Information

Inactivation kinetics of horseradish peroxidase (HRP) by hydrogen peroxide.

ABSTRACT: In recent years, the peroxidase enzymes have generated wide interest in several industrial processes, such as wastewater treatments, food processing, pharmaceuticals, and the production of fine chemicals. However, the low stability of the peroxidases in the presence of hydrogen peroxide (H₂O₂) has limited its commercial use. In the present work, the effect of H₂O₂ on the inactivation of horseradish peroxidase (HRP) was evaluated. Three states of HRP (E₀, E₂, and E₃) were identified. While in the absence of H₂O₂, the resting state E₀ was observed, in the presence of low and high concentrations of H₂O₂, E_2, and E₃ were found, respectively. The results showed that HRP catalyzed the H₂O₂ decomposition, forming the species E_x, which was catalytically inactive. Results suggest that this loss of enzymatic activity is an intrinsic characteristic of the studied HRP. A model from a modified version of the Dunford mechanism of peroxidases was developed, which was validated against experimental data and findings reported by the literature.

SUBMITTER: Morales-Urrea D

PROVIDER: S-EPMC10435507 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Inactivation kinetics of horseradish peroxidase (HRP) by hydrogen peroxide.

Morales-Urrea Diego D López-Córdoba Alex A Contreras Edgardo M EM

Scientific reports 20230817 1

In recent years, the peroxidase enzymes have generated wide interest in several industrial processes, such as wastewater treatments, food processing, pharmaceuticals, and the production of fine chemicals. However, the low stability of the peroxidases in the presence of hydrogen peroxide (H2O2) has limited its commercial use. In the present work, the effect of H2O2 on the inactivation of horseradish peroxidase (HRP) was evaluated. Three states of HRP (E ...[more]

PMID: 37591893

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Project description:Under oxidative stress conditions, mitochondria are the major site for cellular production of reactive oxygen species (ROS) such as superoxide anion and H2O2 that can attack numerous mitochondrial proteins including dihydrolipoamide dehydrogenase (DLDH). While DLDH is known to be vulnerable to oxidative inactivation, the mechanisms have not been clearly elucidated. The present study was therefore designed to investigate the mechanisms of DLDH oxidative inactivation by mitochondrial reactive oxygen species (ROS). Mitochondria, isolated from rat brain, were incubated with mitochondrial respiratory substrates such as pyruvate/malate or succinate in the presence of electron transport chain inhibitors such as rotenone or antimycin A. This is followed by enzyme activity assay and gel-based proteomic analysis. The present study also examined whether ROS-induced DLDH oxidative inactivation could be reversed by reducing reagents such as DTT, cysteine, and glutathione. Results show that DLDH could only be inactivated by complex III- but not complex I-derived ROS; and the accompanying loss of activity due to the inactivation could be restored by cysteine and glutathione, indicating that DLDH oxidative inactivation by complex III-derived ROS was a reversible process. Further studies using catalase indicate that it was H2O2 instead of superoxide anion that was responsible for DLDH inactivation. Moreover, using sulfenic acid-specific labeling techniques in conjunction with two-dimensional Western blot analysis, we show that protein sulfenic acid formation (also known as sulfenation) was associated with the loss of DLDH enzymatic activity observed under our experimental conditions. Additionally, such oxidative modification was shown to be associated with preventing DLDH from further inactivation by the thiol-reactive reagent N-ethylmaleimide. Taken together, the present study provides insights into the mechanisms of DLDH oxidative inactivation by mitochondrial H2O2.

Dataset Information

Inactivation kinetics of horseradish peroxidase (HRP) by hydrogen peroxide.

Publications

Inactivation kinetics of horseradish peroxidase (HRP) by hydrogen peroxide.

Similar Datasets

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