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

CZE has been applied to determination of thermodynamic acidity constants (p K 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, pK a , i mix , of ionogenic groups, and actual ionic mobilities, m i , of gonadotropin‐releasing hormone peptides were determined by nonlinear regression analysis of p H dependence of their effective electrophoretic mobilities. The effective mobilities were measured by CZE in a series of BGE s within a broad p H range (1.80–12.10), at constant ionic strength (25 mM) and reference temperature (25°C). Second, the pK a , i mixvalues were recalculated to thermodynamic p K a s using the D ebye‐ H ückel theory. Thermodynamic p K 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; p K a of imidazolyl group of histidine residues was in the range of 5.7–6.8, p K a of N ‐terminal amino group of the peptide with free N ‐terminus was equal to 6.2, p K a of phenol group of tyrosine residues was in the range of 9.8–10.8, and p K 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 m 2 V −1 s −1 , and ionic mobilities of peptides with charge number ±1 reached values (6.5–12.9) × 10 −9 m 2 V −1 s −1 .