Impact du résidu voisin sur le pKa des peptides et leurs mobilités en électrophorèse capillaire

All theoretical, mathematical and empirical models used to evaluate the electrophoretic mobilities of peptides use three basic parameters: the peptide mass, its number of residues (its length) and its charge. However, all models have a common flaw: the charge parameter (q). When estimating this variable, the use of approximations for the ionization constants (pKa) leads to imprecise values for the calculation. One solution is to limit the development of mobility models to only low pH and assume full protonation of all acidic and basic groups.  In order to understand the effect of the neighboring amino acid (a.a.) residue on the terminal carboxylate and amine functions of a peptide (Cʹ et Nʹ), the pKa values of multiple series of peptides were determined by capillary electrophoresis (CE) using an optimized method. The peptides studied were of the form Z-X and X-Z, where X was one or multiple variable residues and Z was an invariable residue such as phenylalanine, glycine or alanine. The core of this project consisted of determining the actual Cʹ and Nʹ pKa values for 69 peptides to elucidate the effect of their immediate microenvironment. i.e., the effect of their nearest neighbor. This systematic compilation of pKa’s is an approach that is rarely used. With these values on hand, the uncertainty of the ionisation constant that most authors face is almost eliminated and the analysis of the electrophoretic behavior of peptides can be enhanced.  For the peptides studied, we observed that increasing the hydrophobicity of residue X lead to a decrease in Cʹ and Nʹ pKa values, but to different degrees depending on the terminal residue (Z) studied. Hydrophilic X residues possess charged or highly polar side-chains, which influenced Cʹ and Nʹ ionisations to a greater degree than hydrophobic X residues. Additionally, the elongation of a peptide from one to six residues, such as the oligoglycine series studied, lead to an increase in pKa-Cʹ and a decrease in pKa-Nʹ, which might be explained by a diminishing electrostatic interaction between the Cʹ and Nʹ functional groups. These results illustrate that the Cʹ and Nʹ pKa values for peptides are not accurate when based solely on the identity of the Cʹ and Nʹ a.a. residues, which is the classical way peptide charge is estimated for modeling not only mobilities but also chemical and biological reactivity. Potentiometric titration of dipeptides produced pKa values that were compared to pKa’s determined by CE. Significant discrepancies were obtained which brought to light the necessity of rigorously controlling experimental protocols when comparing results obtained with two techniques. Mobility data for high and low pHs were calculated using experimental and literature tabulated pKa values to estimate charge and to therefore understand the benefit gained when applying the Offord mobility model. Finally, simulations of separating 9 peptides by CE were carried out with the computer program PeakMaster and compared with experimental results. This showed the utility and superiority of our cumulated pKa values versus the most widely cited approximate pKa values from the literature.

Keyphrases: pKa, Capillary electrophoresis, Peptide mobility, Peptide charge, Theoretical models, Separation simulation