Human serum albumin modifications associated with reductive radical stress

Radiation-induced modifications of human serum albumin mainly occurring at S-containing residues were investigated by Raman spectroscopy and mass spectrometry techniques. When HO center dot radicals were scavenged by t-BuOH, the H-center dot atom and hydrated electron (e(aq)(-)) attack led to the chemical transformation of Cys into Ala residues and the conversion of Met residues into a-aminobutyric acid residues. Mapping experiments demonstrated that desulfurization selectively affects Cys34, Met123, Met298, Cys514, Met548 and Cys567 since the first stages of reaction. Generation of thiol functionality was also detected at specific residues involved in disulfide bonds, namely Cys200, Cys392 and Cys514, together with partial oxidation at Met87, Met123, Met298, Met329 and Met548, forming sulfoxides. When HO center dot radicals were not scavenged, lesser amounts of the previous modifications were observed, whereas some residues (Cys34, Cys461, Pro486, Phe488 and Phe502) resulted to be oxidatively modified. Based on the known cystine pairing in native albumin, no relationships were observed between desulfurization and disulfide reduction processes at Cys residues involved in disulfide bonds, thus suggesting either independent reactivities or, more probably, reactions at the same amino acids that then underwent quick disulfide scrambling events toward more stable disulfide/thiol populations. When these reactions were performed on protein species added to large unilamellar vesicles, desulfurization yielded sulfur radicals able to induce a cis-trans isomerization of lipids at the onset of irradiation. This study provides a description of the human albumin modifications resulting from reductive radical stress, thus suggesting the need for specific assays and future investigations to detect these events in proteins and lipids within challenged cells.