Determination of acidity constants and ionic mobilities of polyprotic peptide hormones by CZE

CZE has been applied to determination of thermodynamic acidity constants (pK_a) of ionogenic groups and actual ionic mobilities of polyprotic peptides–synthetic human and salmon gonadotropin‐releasing hormones and their derivatives and fragments. First, the mixed acidity constants, , of ionogenic groups, and actual ionic mobilities, m_i, of gonadotropin‐releasing hormone peptides were determined by nonlinear regression analysis of pH dependence of their effective electrophoretic mobilities. The effective mobilities were measured by CZE in a series of BGEs within a broad pH range (1.80–12.10), at constant ionic strength (25 mM) and reference temperature (25°C). Second, the values were recalculated to thermodynamic pK_as using the Debye‐Hückel theory. Thermodynamic pK_a of carboxyl groups was estimated to be in the range of 2.5–3.3 for C‐terminal amino acids of the above peptides, and 5.2 for glutamic acid in the middle of peptide chain; pK_a of imidazolyl group of histidine residues was in the range of 5.7–6.8, pK_a of N‐terminal amino group of the peptide with free N‐terminus was equal to 6.2, pK_a of phenol group of tyrosine residues was in the range of 9.8–10.8, and pK_a of guanidinyl group or arginine residues reached values 11.1–11.3, depending on the position of the residues in the peptide and on the amino acid sequence of the peptide. Absolute values of actual ionic mobilities of peptides with charge number ±2 were in the range (14.6–18.6) × 10⁻⁹ m²V⁻¹s⁻¹, and ionic mobilities of peptides with charge number ±1 reached values (6.5–12.9) × 10⁻⁹ m²V⁻¹s⁻¹.