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

Area of Science:

• Biochemistry
• Oxidative Stress
• Protein Chemistry

Background:

• Singlet molecular oxygen (¹ O₂) plays a role in physiological processes.
• ¹ O₂-mediated protein modifications are important, but its role in crosslinking and aggregation is not well understood.

Purpose of the Study:

• To investigate the involvement of singlet molecular oxygen (¹ O₂) in protein crosslink formation and aggregation using lysozyme as a model.

Main Methods:

• Photochemical oxidation of lysozyme using rose bengal.
• Chemical oxidation using [¹⁸ O]-labeled ¹ O₂ from thermolabile endoperoxides.
• Analysis using SDS-PAGE and nano-scale LC-ESI-MS.

Main Results:

• Both photochemical and chemical oxidation methods induced lysozyme crosslinking and aggregation.
• ¹ O₂ was directly implicated in the formation of covalent crosslinks.
• Crosslinks involved the amino acids histidine, lysine, and tryptophan.

Conclusions:

• Singlet molecular oxygen (¹ O₂) is a direct cause of protein crosslinking and aggregation.
• Identified specific amino acid residues involved in ¹ O₂-induced crosslinking.