文摘
The covalent binding of 4-[13C]- and 5-[13C]-5-chloro-2-methylisothiazol-3-one (MCI) towardhuman serum albumin (HSA) was followed by 13C and 1H{13C} NMR spectroscopy. MCI wasfound to react with histidine through an addition-elimination at position 5, leading to stablesubstitution adducts, and with lysine to form open adducts of the thioamide or amide type. Noother 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 explainedby the rapid reaction of GSH with MCI to form a chlorothioacyl intermediate very reactivetoward primary amino groups of lysine residues. To further confirm these observations andmap covalent binding sites, HSA samples modified by MCI with or without GSH were analyzedby matrix-assisted laser desorption/ionization mass spectrometry of tryptic digests andelectrospray 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 residueswere characterized while when HSA was incubated in the presence of GSH, five peptidesmodified at histidine and lysine residues were identified. These experiments confirmed thatmodifications on lysine residues were of the amide and thioamide types. Observed modificationswere in accordance with mass increases corresponding to structures identified by NMR, andan extra adduct corresponding to a double modification of His 338 was observed. Comparisonof HSA-MCI and HSA-MCI-GSH samples confirmed that the presence of GSH increasedthe modification of lysine residues.