Estimation of acidity constants, ionic mobilities and charges of antimicrobial peptides by capillary electrophoresis.

Abstract

Capillary electrophoresis (CE) was employed for the determination of thermodynamic acidity constants (pKa ) and actual ionic mobilities of polycationic antimicrobial peptides (AMPs). The effective electrophoretic mobilities of AMPs were measured by CE in a series of the background electrolytes within a wide pH range (2.00-12.25), at constant ionic strength (25 mM) and ambient temperature, using polybrene coated fused silica capillaries to suppress sorption of cationic AMPs to the capillary wall. Eventually, Haarhoff-Van der Linde peak fitting function was used for the determination of correct migration times of some AMPs peaks that were distorted by electromigration dispersion. The measured effective mobilities were corrected to 25°C. Mixed acidity constants, pKa,i mix , and actual ionic mobilities, mi , of AMPs were determined by the nonlinear regression analysis of pH dependence of their effective mobilities. The pKa,i mix values were recalculated to thermodynamic pKa s using the Debye-Hückel theory. Thermodynamic pKa of imidazolium group of histidine residues was found to be in the range 3.72-4.98, pKa of α-NH3+ group was in the range 6.14-6.93, and pKa of ε-NH3+ group of lysine spanned the interval 7.26-9.84, depending on the particular amino acid sequence of the AMPs. Actual ionic mobilities of AMPs with positive charges from one to six elementary units achieved values (9.8 - 36.5) × 10-9 m2 V-1 s-1 .