Study of Lysozyme Glycation Reaction by Mass Spectrometry and NMR Spectroscopy

The Advanced Glycation End Products (AGEs) are the causative substances of lifestyle‐habit illness. To elucidate the glycation mechanism of the protein, the reaction of lysozyme with D ‐glucose was analyzed by the fluorescence, TOF‐MS, and ¹³C‐NMR spectroscopy under the physiological condition. The fluorescence intensity of lysozyme in the glycation solution increased proportionally with a reaction time of ten weeks. The MALDI‐TOF‐MS spectra of the reaction solution after two weeks showed a peak at m/z 15066, which indicated the presence of a larger molecule than the native lysozyme (m/z 14331), and new peaks at m/z 30105 (dimer) and 45000 (trimer) were also observed. The spectral analysis supported the assumption of a continuous glycation reaction of D ‐glucose with lysozyme and a 30% transformation of lysozyme to the dimeric form during ten weeks. The ¹³C‐NMR spectra of lysozyme showed six ¹³C]‐labeled signals by the glycation reaction with ¹³C]‐glucose after two weeks of reaction. The combined analysis of TOF‐MS and ¹³C‐NMR spectra uncovered that first products of the glycation reaction of lysozyme with D ‐glucose can be observed already three hours after starting the reaction and that nine D ‐glucose units are attached during ten weeks at 37°.