Studies on Non-covalent Complexes of Glutathione with L-Aromatic Amino Acids by Electrospray Ionization Mass Spectrometry

To explore the non-covalent interaction between reduced tripeptide glutathione and L-aromatic amino acids, a stoichiometry of reduced gamma-glutathione and three L-aromatic amino acids, including phenylalanine, tyrosine and tryptophan were mixed respectively, and then incubated at room temperature and physiological pH conditions for 1 h to reach the equilibrium. The electrospray ionization mass spectrometry (ESI-MS) results indicate that glutathione and three L-aromatic amino acids could form non-covalent complexes, respectively. The primary fragment ions of complexes obtained from MS2 in a tandem mass spectrometer included aromatic amino acid, glutathione as well as its y(2), b(2), which confirmed the results of ESI-MS. Moreover, in UV spectroscopies, the discrepancy between complexes and reactants also confirmed the formation of complexes in aqueous solution. To avoid distinct ionization efficiency discrepancy and signal suppression in ESI-MS measurements, the interaction between glutathione and L-Tyrosine was evaluated quantitatively, which revealed that the reactant concentration should range from 5 x 10(-5) to 3.00 x 10(-4) mol/L. The dissociation constants of three complexes were determined using mass spectrometric titration method, the calculation results revealed that the stabilities of complexes formed by glutathione and three L-aromatic amino acids increased gradually according to the order of tyrosine, tryptophan and phenylalanine.