Study of Lysozyme Glycation Reaction by Mass Spectrometry and NMR Spectroscopy |
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Authors: | Yukiko Maekawa Makiko Sugiura Atsuko Takeuchi Koji Tomoo Toshimasa Ishida Miyoko Kamigauchi |
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Affiliation: | 1. Department of Physical Chemistry, Kobe Pharmaceutical University, 4‐19‐1 Motoyama‐kitamachi, Higashinada‐ku, Kobe 658‐8558, Japan (phone: +81‐78‐441‐7540;2. fax: +81‐78‐441‐7541);3. Osaka University of Pharmaceutical Sciences, 4‐20‐1 Nasahara, Takatsuki Osaka 569‐1094, Japan |
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Abstract: | 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°. |
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Keywords: | Lysozymes Glycation Advanced glycation end products (AGEs) Mass spectrometry NMR Spectroscopy |
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