Figure 5.

pH titration curves of 3F4HPA in the absence of WT C₂ at 25 °C. A, pH dependence of the ¹⁹F chemical shift of 3F4HPA. Fitting to the Henderson-Hasselbalch equation (Equation 3) yielded pK_a values of 4.2 ± 0.04 and 9.0 ± 0.02 for the carboxyl and hydroxyl group, respectively. Structures of the ionization states of 3F4HPA dominating in the three pH regions are shown, based on the data in B. B, ¹³C NMR determination of the events responsible for the different pK_a values. The carboxyl carbon near 180 ppm is most sensitive to the pK_a of 4.2, and the phenolic carbon near 140 ppm (at pH 7.0) is most sensitive to the pK_a of 9.0.