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Characterisation of peroxidasin activity in isolated extracellular matrix and direct detection of hypobromous acid formation
- Bathish, Boushra, Turner, Rufus, Paumann-Page, Martina, Kettle, Anthony J., Winterbourn, Christine C.
- Archives of biochemistry and biophysics 2018 v.646 pp. 120-127
- acetaminophen, bromides, catalase, collagen, crosslinking, enzyme activity, enzyme inhibitors, epithelial cells, extracellular matrix, hydrazides, hydrogen peroxide, hydroxylysine, mass spectrometry, methionine, myeloperoxidase, oxidation, peroxidase, thiocyanates
- Peroxidasin is a heme peroxidase that catalyses the oxidation of bromide by hydrogen peroxide to form an essential sulfilimine cross-link between methionine and hydroxylysine residues in collagen IV. We investigated cross-linking by peroxidasin embedded in extracellular matrix isolated from cultured epithelial cells and its sensitivity to alternative substrates and peroxidase inhibitors. Peroxidasin showed peroxidase activity as measured with hydrogen peroxide and Amplex red. Using a specific mass spectrometry assay that measures NADH bromohydrin, we showed definitively that the enzyme releases hypobromous acid (HOBr). Less than 1 μM of the added hydrogen peroxide was used by peroxidasin. The remainder was consumed by catalase activity that was associated with the matrix. Results from NADH bromohydrin measurements indicates that low micromolar HOBr generated by peroxidasin was sufficient for maximum sulfilimine cross-linking, whereas 100 μM reagent HOBr or taurine bromamine was less efficient. This implies selectivity for the enzymatic process. Physiological concentrations of thiocyanate and urate partially inhibited cross-link formation. 4-Aminobenzoic acid hydrazide, a commonly used myeloperoxidase inhibitor, also inhibited peroxidasin, whereas acetaminophen and a 2-thioxanthine were much less effective. In conclusion, HOBr is produced by peroxidasin in the extracellular matrix. It appears to be directed at the site of collagen IV sulfilimine formation but the released HOBr may also undergo other reactions.