Figure 5.
Product formation of the reaction of vCPH with three peptide substrates. Plots of the reaction of vCPH in complex with Fe(II), 2-OG, and peptide with oxygen are shown. Three peptide substrates were assayed and the samples chemically quenched with 1% TFA at different time points. Quenched samples were analyzed to determine the percentage of peptide hydroxylation using eq 1 (A) and levels of succinate (B) during the reaction. The data were fitted to a single-exponential function shown as a red line eq 3. Final conditions: 800 μM vCPH, 800 μM FeSO4, 1 mM 2-OG, and 1 mM peptide in 100 mM HEPES (pH 7.5) under anaerobic conditions at 5 °C, reacted with O2-saturated buffer.