GSH substitutes for vitamin C as a cofactor in HIF-1α hydroxylation in vitro. (A) GSH can enhance PHD hydroxylation activity in the absence of ascorbate (−Asc.) in a dose-dependent manner. Hydroxylation activity was determined using a multiwell VBC binding assay. (B) PHD-dependent hydroxylation reaction rate in the presence of 2mM ascorbate (control) or 2mM ascorbate combined with 5mM GSH (+5mM GSH). Shown are mean values ± SEM of triplicates. (C) Ascorbate determination by HPLC before and after 1 hour of PHD2-dependent hydroxylation reaction (left panels). Ascorbate content is only slightly decreased after 1 hour of incubation and independent of target hydroxylation (right panel). Shown are mean values ± SEM of 3 independent experiments normalized to values measured at time point zero.