Effect of glutathione on the covalent binding of the 13C-labeled skin sensitizer 5-chloro-2-methylisothiazol-3-one to human serum albumin:: Identification of adducts by nuclear magnetlic resonance, matrix-assisted laser desorption/ionization mass spectrometry, and nanoelectrospray tandem mass spectrometry

The covalent binding of 4-[C-13]- and 5-[C-13]-5-chloro-2-methylisothiazol-3-one (MCI) toward human serum albumin (HSA) was followed by C-13 and H-1{C-13} NMR spectroscopy. MCI was found to react with histidine through an addition-elimination at position 5, leading to stable substitution adducts, and with lysine to form open adducts of the thioamide or amide type. No other modification could be detected on either cysteine or tyrosine. In the presence of glutathione (GSH), we observed an increased covalent binding to lysine residues. This could be explained by the rapid reaction of GSH with MCI to form a chlorothioacyl intermediate very reactive toward primary amino groups of lysine residues. To further confirm these observations and map covalent binding sites, HSA samples modified by MCI with or without GSH were analyzed by matrix-assisted laser desorption/ionization mass spectrometry of tryptic digests and electrospray tandem mass spectrometry of modified peptides purified by reverse phase HPLC. About 80% of the HSA sequence was mapped, and several modified peptides were identified. When HSA was incubated with MCI without GSH, three peptides modified at histidine residues were characterized while when HSA was incubated in the presence of GSH, five peptides modified at histidine and lysine residues were identified. These experiments confirmed that modifications on lysine residues were of the amide and thioamide types. Observed modifications were in accordance with mass increases corresponding to structures identified by NMR, and an extra adduct corresponding to a double modification of His 338 was observed. Comparison of HSA-MCI and HSA-MCI-GSH samples confirmed that the presence of GSH increased the modification of lysine residues.