Figure 4.

Glutathione interferes with AGE formation even in the absence of DJ-1. (A) To determine the influence of free thiols on glycation reactions, 1 mM peptide 1 (Ac-LESRHYA) was allowed to react with 1 mM MGO and was then analyzed by LC-MS after subsequent or concurrent treatment with DJ-1^WT and/or glutathione (GSH) in 20 mM phosphate buffer at pH 7.3 for 3 h at 37 °C. Distribution of glycation adducts observed by LC-MS after (B) subsequent or (C) concurrent DJ-1 and/or GSH treatment. Decreases in glycation were only observed in concurrent, not subsequent, conditions. While the addition of either GSH or DJ-1^WT was able to reduce glycation during concurrent incubations, there was also an additive effect where even greater reductions in glycation were observed in the presence of both GSH and DJ-1^WT. Stacked bar graphs are plotted as mean ± standard deviation for each mass adduct. A nondirectional (two-tailed) one-way ANOVA using Dunnett’s multiple comparison test was used to determine statistically significant differences in total glycation, p < 0.0001(****) and p < 0.001(***).