1H NMR Spectroscopy As Tool To Study Transglutaminase Crosslinking Of Pea Globulin

Abstract : A new method based on NMR spectroscopy was developed to detect the G-L (GlutamylLysine) isopeptide bonds formed by the enzymatic transglutaminase reaction. Because of the complexity of NMR signals of proteins due to their structures, the method was first developed on a model system (glutamine and lysine) to simplify the detection of the G-L residue. And then, the results were applied on the real protein matrix (pea globulin). MTG treatment of model system led to the appearance of a new resonance on NMR spectrum which is originated probably from the ε-methylene protons of lysine residues covalently linked to glutamine. The comparison between NMR spectra of MTG-treated and untreated pea globulin with MTG-treated model system permitted to identify the G-L isopeptide signal. The G-L isopeptide signal is also observed in the NMR spectra of native pea globulin (untreated which MTG), indicating that the isopeptide is naturally present in pea proteins. However, the superimposition of NMR spectra of MTG-treated and untreated pea globulin shows a higher intensity of G-L isopeptide signal for MTG-treated samples. The increased signal intensity evidences the enzymatic reaction and permits to quantify the G-L isopeptide.