Fig. 6 | Scientific Reports

Fig. 6

From: Human prolyl hydroxylase domain 2 reacts with O2 and 2-oxoglutarate to enable formation of inactive Fe(III).2OG.hypoxia-inducible-factor α complexes

Fig. 6

Hydroxylation assays and UV-vis spectra showing that L-Asc, but not thiol reducing agents, hinders formation of the ∼600 nm absorbance. (a) Comparison of hydroxylation observed when a L-Asc and PHD2.Fe(II).2OG mixture (green bars) is added to HIF1α-CODD556 − 574 either after O2 exposure (blue bars), or before O2 exposure (orange bars) (see Experimental Procedure for details). No activity loss was observed for the anaerobic and L-Asc containing samples (orange and green bars). The results are means of 3 independent runs (n = 3, mean ± SD). (b-e) UV-vis spectra of 1 mM L-Asc/D-isoascorbic acid/DTT/GSH and PHD2.Fe(II).2OG mixture analysed after 0 (purple), 30 (green), 60 (orange) and 120 min (light blue) of O2 exposure. The blue chromophore was not observed in the L-Asc containing sample. IAA was also observed to slow formation of the blue complex. No clear effect was observed with DTT. An increase in rate of formation of the blue chromophore was observed with GSH.

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