2.3. pKa Determination by Potentiometric Titration

Potentiometric titration for the determination of the acid dissociation constant (pKa) values for 2-oxo-IDPs and IDPs was performed using a F-51 pH meter (HORIBA, Kyoto, Japan) combined with a 9618S-10D Micro ToupH electrode (HORIBA) as described previously [23], with slight modifications. The pH meter was calibrated with standard buffers (HORIBA), i.e., pH 4.01 ± 0.02 (50 mM potassium hydrogen phthalate), pH 6.86 ± 0.02 (25 mM potassium dihydrogenphosphate-dipotassium hydrogenphosphate), and pH 9.18 ± 0.02 (10 mM sodium tetraborate). 2-oxo-IDPs and IDPs were dissolved in ultrapure water and their pH adjusted to 2.0 with 1 M HCl. In brief, the dipeptide solutions (10 mM, 1 mL) were titrated potentiometrically with a dropwise addition of 1 M NaOH at room temperature (25 °C). Sufficient time (about 10–15 s) was allowed to obtain a reasonably stable pH reading before the next base addition. The dipeptide solutions were completely mixed during potentiometric titration with a magnetic stirrer. The pKa values were calculated by the second-derivative plot as described previously [23], and are summarized in Supplementary Table S1.