Fig. 2. (A) Effect of Fe²⁺ on the rate of prolyl hydroxylase (PHD2) reaction at different concentrations of 2-oxoglutarate (2-OG). Iron binds uncompetitively to PHD2, as indicated by lines that will intersect in the double reciprocal plot. (B) The double reciprocal plot of ascorbate concentrations at different levels of 2-OG show parallel lines above 1/[Asc]₀=0.01 µM, while at higher ascorbate concentrations the lines begin to converge. This indicates ascorbate predominantly reacting with Fe³⁺, and not binding to the hydroxylases, at low ascorbate concentrations; at higher ascorbate levels, there is significant ascorbate reacting in iron reduction and the overall hydroxylation reaction. (C) The effect of ascorbate on the hydroxylation reaction. When there is no ascorbate, the reaction occurs though it takes more than 30 minutes for HIF1 to be fully degraded.