Exploring the evaluation of net charge, hydrodynamic size and shape of peptides through experimental electrophoretic mobilities obtained from CZE

Abstract

This work explores the validity of simple CZE models to analyze the electrophoretic mobilities of 102 peptides reported in literature. These models are based mainly on fundamental physicochemical theories providing analytical expressions amenable to relatively simple numerical analysis. Thus, the Linderstrøm-Lang capillary electrophoresis model (LLCEM) and its perturbed version (PLLCEM), proposed and applied previously to the CZE of globular proteins, are adapted and used here for peptides. Also the effects of pK-shifts on net charge, hydration and hydrodynamic size and shape of peptides are analyzed and discussed. Emphasis is placed on the fact that these parameters are physically coupled, and thus a variation in the net charge may produce an appreciable change in the hydrodynamic size of peptides. Within the framework of CZE, peptides may be assumed as having a hydrodynamic volume associated with either spherical or spheroidal particles. The effects on peptide net charge and hydrodynamic size, of electrostatic interaction between a pair of charged groups in the chain and electrical permitivitty around the peptide domain are studied. The predictions of the PLLCEM and LLCEM are in good agreement with results reported previously in the literature. Several limitations concerning these models and some needs for further research are also described.

Analysis of the interplay among charge, hydration and shape of proteins through the modeling of their CZE mobility data

“…Thus, the relevant variable is the equivalent Electrophoresis 2009Electrophoresis , 30, 2328Electrophoresis -2336 sphere with a Stokes equivalent hydrodynamic radius a e H , which must be defined in terms of the main particle dimensions, as shown below. Specific discussions concerning the physical significance of each term in Equations (1) and (2), may be found elsewhere [15,16,22]. For calculation purposes, the expression [15] and its addendum for a detailed discussion on this expression).…”

“…Here, in the PLLCEM the equivalent spherical model (ESM) of CZE is used again, which has been illustrated in [16,17,22] and validated in particular for spheroidal particles of low eccentricity, usually selected as prototype hydrodynamic shapes for proteins and peptides. The ESM has also been used widely in the literature and in basic texts to estimate diffusion and sedimentation coefficients [32,33].…”

“…Also activity coefficients g i are included as indicated in [17]. The evaluation of protein-effective charge requires the knowledge of actual pK i values of ionizing groups of side chains and ionizing terminal -COOH and -NH 2 groups [15,16,22,28]. The pK i values are mainly a result of electrostatic interactions among protein ionizing groups and ions in the BGE, yielding a shift DpK i of the reference pK r i , which in turn refers to the hypothetical pK value where one assumes that all other titrating sites in the analyte are fixed in their electrically neutral state (see [22] for a discussion concerning pK r i and also Section 3).…”

“…One model type of interest is that considering the ''inverse problem'' [17] where, for a given protocol involving wellspecified bulk pH, ionic strength I, temperature T, electrical permittivity e and viscosity Z of the BGE, the experimental effective mobility is provided as the basic data to evaluate analyte properties such as, for instance, hydration, effective electrical charge, hydrodynamic size and shape, and pH-microenvironment, among others. This basic problem has associated the charge regulation phenomenon, also designated proton-binding cooperativity, which is always present around electrophoretically migrating particles [15][16][17][18][19][20][21][22][23][24]. This phenomenon is relevant to estimate the protein pK-shifts of the i-ionizing group designated DpK i , the pH near molecule pH Ã , and the pH near the i-ionizing group indicated as pH i , for i 5 1yN c .…”

“…In particular, relevant reviews including CZE of proteins and peptides are available (see, for instance, [2][3][4][5] and citations therein). At present CZE models are useful in the physicochemical characterization and interpretation of the effective mobility data of peptides and proteins [6][7][8][9][10][11][12][13][14][15][16]. One model type of interest is that considering the ''inverse problem'' [17] where, for a given protocol involving wellspecified bulk pH, ionic strength I, temperature T, electrical permittivity e and viscosity Z of the BGE, the experimental effective mobility is provided as the basic data to evaluate analyte properties such as, for instance, hydration, effective electrical charge, hydrodynamic size and shape, and pH-microenvironment, among others.…”