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 (pK ) 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, m , 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 pK s using the Debye-Hückel theory. Thermodynamic pK of imidazolium group of histidine residues was found to be in the range 3.72-4.98, pK of α-NH group was in the range 6.14-6.93, and pK of ε-NH 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 m V s .

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

“…Electromigration behavior of polycationic antimicrobial peptides (AMBPs) in acidic, neutral, and basic aqueous solutions was studied by their CZE analyses in the BGEs at wide pH range 2.00–12.25 at constant ionic strength 25 mM and ambient temperature using polybrene‐coated fused silica (FS) capillaries to suppress sorption of strongly basic AMBPs to the capillary wall . The measured effective mobilities were corrected to 25°C.…”

“…Effective mobilities of polyprotic peptides–synthetic insect polycationic antimicrobial peptides and their derivatives and fragments were measured by CZE within a wide pH range 2.00–12.25 in aqueous BGEs at constant ionic strength 25 mM and at ambient temperature . From the pH dependence of effective mobilities (corrected to 25°C), first the mixed acidity constants, p K a,mix , of ionogenic groups and ionic mobilities of particular ionic forms of these peptides were obtained by nonlinear regression analysis.…”

Determination of thermodynamic acidity constants and limiting ionic mobilities of weak electrolytes by capillary electrophoresis using a new free software AnglerFish

“…There were many attempts to use CZE to determine both pKa and ionic mobilities, see, for example, Refs. []. Šlampová et al.…”

Determination of physicochemical parameters of (bio)molecules and (bio)particles by capillary electromigration methods

“…of water-insoluble or sparingly soluble compounds, hydro-organic or organic solvents can be utilized. To accelerate pK a determination, various strategies, such as short-end injection [119], pressure-assisted CE [120,121], dynamically coated capillaries [122][123][124], or multiplexed CE [125,126], have been used.…”

“…Acidity constants of non‐UV absorbing compounds can be determined by combining CE with indirect UV‐detection, contactless conductivity detection [109, 118], amperometric detection, or MS. To determine acidity constants of water‐insoluble or sparingly soluble compounds, hydro‐organic or organic solvents can be utilized. To accelerate p K a determination, various strategies, such as short‐end injection [119], pressure‐assisted CE [120, 121], dynamically coated capillaries [122–124], or multiplexed CE [125, 126], have been used.…”