Figure 4.
Recombinant PHD2181-402 expression and Michaelis-Menten constant (Km) determination of 2-OG for PHD2181-402. (a) PHD2181-402 compared to PHD1 (407 amino acid residues), PHD2 (426), and PHD3 (239), showing overall homology between the three human PHDs and PHD2181-402 spanning the highly conserved active site. Boxes indicate expressed amino acids, and overlap between boxes indicates amino acid sequence homology. Percent identity (BLAST) is indicated for each PHD isoform in comparison to PHD2181-402. (b) Size exclusion superdex 75 column elution of PHD2181-402. (Cond: conductivity (mS/cm), concB: percentage of buffer B used). (c) 12% SDS gel image of the Superdex 75 column eluent confirming the expression and purity of PHD2181-402. (d) 100 nM of PHD2181-402 enzyme was incubated with various concentrations of substrate 2-OG from 625 nM to 40 μM,10 μM HIF-1α547-581 peptide, 10 μM Fe (II), and 100 μM ascorbic acid and concentration of product succinate was measured after a 10-minute reaction to determine the rate (n = 4, error bars: SEM, Michaelis-Menten least squares fit)