Novel Intra- and Inter-molecular Sulfinamide Bonds in S100A8 Produced by Hypochlorite Oxidation

Publication Type:
Journal Article
Journal of Biological Chemistry, 2001, 276 (36), pp. 33393 - 33401
Issue Date:
Full metadata record
Files in This Item:
Filename Description Size
Thumbnail2006009317OK.pdf369.51 kB
Adobe PDF
Hypochlorite is a major oxidant generated when neutrophils and macrophages are activated at inflammatory sites, such as in atherosclerotic lesions. Murine S100A8 (A8) is a major cytoplasmic protein in neutrophils and is secreted by macrophages in response to inflammatory stimuli. After incubation with reagent HOCl for 10 min, ∼85% of A8 was converted to 4 oxidation products, with electrospay ionization mass spectrometry masses of m/z 10354, 10388, 10354 ± 1, and 20707 ± 3. All were resistant to reduction by dithiothreitol. Initial formation of a reactive Cys sulfenic acid intermediate was demonstrated by the rapid conjugation of 5,5-dimethyl-1,3-cyclohexanedione (dimedone) to HOCl-treated A8 to form stable adducts. Matrix-assisted laser desorption-reflectron time of flight peptide mass fingerprinting of isolated oxidation products confirmed the mass additions observed in the full-length proteins. Both Met36/73 were converted to Met36/73 sulfoxides. An additional product with an unusual mass addition of m/z 14 (±0.2) was identified and corresponded to the addition of oxygen to Cys41, conjugation to various ε-amines of Lys6, Lys34/35, or Lys87 with loss of dihydrogen and formation of stable intra- or inter-molecular sulfinamide cross-links. Specific fragmentations identified in matrix-assisted laser desorption-post source decay spectra and low energy collisional-induced dissociation tandem mass spectroscopy spectra of sulfinamide-containing digest peptides confirmed Lys34/35 to Cys41 sulfinamide bonds. HOCl oxidation of mutants lacking Cys41 (Ala41S100A8) or specific Lys residues (e.g. Lys34/35, Ala34/35S100A8) did not form sulfinamide cross-links. HOCl generated by myeloperoxidase and H 2O2 and by phorbol 12-myristate 13-acetate-activated neutrophils also formed these products In contrast to the disulfide-linked dimer, oxidized monomer retained normal chemotactic activity for neutrophils. Sulfinamide bond formation represents a novel oxidative cross-linking process between thiols and amines and may be a general consequence of HOCl protein oxidation in inflammation not identified previously. Similar modifications in other proteins could potentially regulate normal and pathological processes during aging, atherogenesis, fibrosis, and neurogenerative diseases.
Please use this identifier to cite or link to this item: