Singlet oxygen-induced protein aggregation: Lysozyme crosslink formation and nLC-MS/MS characterization

Abstract

Singlet molecular oxygen (¹ O₂ ) has been associated with a number of physiological processes. Despite the recognized importance of ¹ O₂ -mediated protein modifications, little is known about the role of this oxidant in crosslink formation and protein aggregation. Thus, using lysozyme as a model, the present study sought to investigate the involvement of ¹ O₂ in crosslink formation. Lysozyme was photochemically oxidized in the presence of rose bengal or chemically oxidized using [¹⁸ O]-labeled ¹ O₂ released from thermolabile endoperoxides. It was concluded that both ¹ O₂ generating systems induce lysozyme crosslinking and aggregation. Using SDS-PAGE and nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry, the results clearly demonstrated that ¹ O₂ is directly involved in the formation of covalent crosslinks involving the amino acids histidine, lysine, and tryptophan.

Keywords: crosslink; histidine; protein; singlet molecular oxygen; tryptophan.