Mechanism and stoichiometry of 2,2-diphenyl-1-picrylhydrazyl radical scavenging by glutathione and its novel α-glutamyl derivative

Kinetic mechanism and stoichiometry of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical by glutathione and its novel analog, containing α-glutamyl residue in place of the γ-glutamyl moiety, were studied using different ratios of reagents. At low concentrations of the peptides, the process was described as a bimolecular reaction obeying the stoichiometric ratio 1:1. However, at excess of peptides the formation of a non-covalent complex between the reagents was discovered and characterized by dissociation constants K = 0.61 mM for glutathione and K = 0.27 mM for the glutathione α-glutamyl analog, respectively. The complex formation was followed by a reaction step that was characterized by the similar rate constant k = 0.02 s−1 for both peptides. Thus, the apparently different antioxidant activity of these two peptides, observed under common assay conditions, was determined by differences in the formation of this non-covalent complex.

Graphical abstract2,2-Diphenyl-1-picrylhydrazyl radical (DPPH) forms a non-covalent complex with glutathione (left) and its novel α-glutamyl analog (right) and this equilibrium determines the apparently different antioxidant activity of these peptides in vitro assay with DPPH.Figure optionsDownload full-size imageDownload as PowerPoint slide