Lysozyme fragmentation induced by gamma-radiolysis

Area of Science:

• Biochemistry
• Protein Chemistry
• Radiation Chemistry

Background:

• Lysozyme is a model protein extensively studied for its structural and functional properties.
• Understanding protein radiolysis is crucial for applications in food science, medicine, and radiation biology.
• Previous studies have indicated protein fragmentation upon irradiation, but precise site localization remains challenging.

Purpose of the Study:

• To precisely map the radio-fragmentation sites on the lysozyme backbone induced by irradiation in frozen states.
• To elucidate the mechanism of peptide bond cleavage under these specific conditions.
• To correlate fragmentation sites with the three-dimensional structure of lysozyme.

Main Methods:

• Irradiation of lysozyme in frozen states under anoxic conditions.
• Separation of radiolysis fragments using SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
• N-terminal sequencing of fragments to identify amino acid sequences.
• Purification of fragments using reverse-phase high-performance liquid chromatography (RP-HPLC).
• Molecular mass determination using electro-spray ionization mass spectrometry (ESI-MS).
• Total amino acid analysis for comprehensive characterization.

Main Results:

• Specific fragmentation sites were identified along the lysozyme backbone.
• Radio-fragmentation predominantly occurred at solvent-exposed loops and turns on the protein surface.
• Evidence confirmed the direct breakage of the peptide bond (CO-NH).
• The combination of techniques provided high confidence in site localization and fragment characterization.

Conclusions:

• Irradiation of frozen lysozyme under anoxic conditions leads to site-specific peptide bond cleavage.
• The location of fragmentation sites suggests a susceptibility of surface-exposed regions to radiation damage.
• This study provides detailed insights into the molecular mechanisms of protein radiolysis.