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EPR spin-trapping of a myeloperoxidase protein radical
O M Lardinois1, P R Ortiz de Montellano
1Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446, USA.
Abstract:
Incubation of myeloperoxidase (MPO) with H(2)O(2) in the presence of the spin trap DBNBS (3,5-dibromo-4-nitrosobenzenesulfonic acid) results in the EPR-detectable formation of a partially immobilized protein radical. The radical was only formed in the presence of both MPO and H(2)O(2), indicating that catalytic turnover of the protein is required. The changes in the EPR spectrum of the adduct upon treatment with pronase confirm that the spin trap is bound to a protein residue. These results establish that MPO, like lactoperoxidase [Lardinois, O. M., Medzihradszky, K. F., and Ortiz de Montellano, P. R. (1999) J. Biol. Chem. 274, 35441-35448], reacts with H(2)O(2) to give a protein radical intermediate. The protein radical may have a catalytic role, may be related to covalent binding of the prosthetic heme group to the protein, or may reflect a process that leads to inactivation of the enzyme.
Insights
Myeloperoxidase (MPO) forms a detectable protein radical when reacting with hydrogen peroxide (H2O2), indicating catalytic turnover is necessary. This radical may play a role in enzyme function or inactivation.
Area of Science:
- Biochemistry
- Enzymology
- Free Radical Chemistry
Background:
- Myeloperoxidase (MPO) is a key enzyme in the innate immune system.
- Understanding MPO's catalytic mechanism is crucial for its role in inflammation and host defense.
- Previous studies have shown similar radical intermediates in other peroxidases.
Purpose of the Study:
- To investigate the formation and nature of reactive intermediates during MPO catalysis.
- To determine if MPO generates protein-based radicals upon reaction with hydrogen peroxide.
- To explore the potential roles of these radicals in MPO's enzymatic activity.
Main Methods:
- Incubation of MPO with hydrogen peroxide (H2O2) and the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS).
- Electron Paramagnetic Resonance (EPR) spectroscopy to detect and characterize the protein radical.
- Pronase treatment to confirm spin trap binding to protein residues.
Main Results:
- EPR-detectable formation of a partially immobilized protein radical was observed.
- Radical formation required both MPO and H2O2, indicating catalytic turnover.
- Pronase treatment confirmed the spin trap's covalent attachment to a protein residue.
- MPO, similar to lactoperoxidase, generates a protein radical intermediate with H2O2.
Conclusions:
- MPO catalyzes the formation of a protein radical intermediate during its reaction with H2O2.
- This protein radical may be involved in MPO's catalytic cycle, heme binding, or enzyme inactivation.
- Further research is needed to elucidate the precise role of this radical intermediate.