Figure 3.

Chemical shift titrations of the carboxylate groups of Glu¹¹ and Asp¹⁹. (A) Superimposed small regions of cross sections taken through the 3D HCCO spectra taken over the range from pH 6.5 to 2.9, showing the correlation between the carboxylic acid ¹³C and its vicinal methylene ¹H chemical shifts. Chemical shift versus pH for (B) the Glu¹¹ side chain ¹³C^δ (black) and ¹³C^γ (red), and (C) the Asp¹⁹ side chain ¹³C^γ (black) and ¹³C^β (red) are fit to the Henderson-Hasselbalch equation using non-linear least-squares regression. Fitted values for (B) are: pK = 5.31±0.01, with ¹³C^δ and ¹³C^γ chemical shift of 179.2 ppm and 33.0 ppm, respectively, for the protonated form (COOH), and a ¹³C^δ and ¹³C^γ chemical shift of 183.2 ppm and 36.3 ppm for the deprotonated form (COO⁻). Fitted values for Asp¹⁹ are: pK = 4.35±0.02, with ¹³C^γ and ¹³C^β chemical shift of 175.5 ppm and 37.1 ppm, respectively, for the protonated form, and ¹³C^γ and ¹³C^β chemical shifts of 178.8 ppm and 40.3 ppm for the deprotonated form (COO⁻).