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Updated: Aug 12, 2025
11:56
A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
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Autoxidation of ascorbate mediates lysine N-pyrrolation
Jun Yoshitake1, Takahiro Shibata1,2, Miho Chikazawa3
1Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya University, Nagoya, Japan.
Free Radical Research
|February 1, 2023
Summary
Protein N-pyrrolation creates Nε-pyrrole-l-lysine (pyrK) residues, which mimic DNA and are implicated in autoimmune responses. Ascorbate oxidation, like lipid peroxidation, generates these pyrK modifications, suggesting a role in innate and autoimmune diseases.
Area of Science:
- Biochemistry
- Immunology
- Oxidative Stress
Background:
- Protein N-pyrrolation is a natural modification converting lysine to Nε-pyrrole-l-lysine (pyrK).
- Pyrrolated proteins bind DNA-staining agents and antibodies, acting as DNA mimics.
- These properties link pyrK to innate and autoimmune responses, with lipid peroxidation as a suspected source.
Purpose of the Study:
- To investigate if metal-catalyzed ascorbate oxidation also induces protein N-pyrrolation.
- To identify pyrK formation factors during ascorbate autoxidation.
- To explore the role of ascorbate-derived pyrK in autoimmune responses.
Main Methods:
- Incubation of human serum albumin (HSA) with Fe2+/ascorbate.
- Detection of N-pyrrolation using SYBR Green I (SG) staining and anti-DNA antibodies.
- Analysis of pyrK formation in hemoglobin and identification of pyrrolation factors.
Main Results:
- Fe2+/ascorbate treatment converted HSA to SG-binding protein, indicating N-pyrrolation, even without polyunsaturated fatty acids.
- PyrK formation was confirmed in ascorbate-treated HSA and hemoglobin.
- Glycolaldehyde and glyoxal were identified as pyrrolation factors from ascorbate oxidation.
- Sera from MRL-lpr mice recognized proteins modified by Fe2+/ascorbate and aldehydes.
Conclusions:
- Metal-catalyzed ascorbate oxidation, similar to lipid peroxidation, generates N-pyrrolated proteins (pyrK).
- Ascorbate autoxidation can be an endogenous source of autoantigenic pyrK proteins.
- PyrK residues may contribute to innate and autoimmune responses linked to ascorbate's oxidative metabolism.
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