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 the fluorescence, TOF-MS, and C-13-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 week showed a peak and m/z 15066, which indicated the presence of a larger molecule than the native lysozyme (m/z 143312), and new peaks at m/z 30105 (dimer) and (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 C-13-NMR spectra of lysozyme showed six[C-13]-labeled signals by the glycation reaction with [C-13]-glucose after two weeks of reaction. The combined analysis of TOF-MS and C-13-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 degrees.