Determination of the microenvironment‐pH and charge and size characteristics of amino acids through their electrophoretic mobilities determined by CZE

Abstract

Effective electrophoretic mobility data of 20 amino acids reported in the literature are analyzed and interpreted through simple physicochemical models, which are able to provide estimates of coupled quantities like hydrodynamic shape factor, equivalent hydrodynamic radius (size), net charge, actual pK values of ionizing groups, partial charges of ionizing groups, hydration number, and pH near molecule (microenvironment-pH of the BGE). It is concluded that the modeling of the electrophoretic mobility of these analytes requires a careful consideration of hydrodynamic shape coupled to hydration. In the low range of pH studied here, distinctive hydrodynamic behaviors of amino acids are found. For instance, amino acids with basic polar and ionizing side chain remain with prolate shape for pH values varying from 1.99 to 3.2. It is evident that as the pH increases from low values, amino acids get higher hydrations as a consequence each analyte total charge also increases. This result is consistent with the monotonic increase of the hydrodynamic radius, which accounts for both the analyte and the quite immobilized water molecules defining the electrophoretic kinematical unit. It is also found that the actual or effective pK value of the alpha-carboxylic ionizing group of amino acids increases when the pH is changed from 1.99 to 3.2. Several limitations concerning the simple modeling of the electrophoretic mobility of amino acids are presented for further research.

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

“…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].…”

“…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). Thus, the pHmicroenvironment around the i-ionizing group, designated pH i , is a function of physicochemical properties of the electrolyte solution surrounding the macromolecule, and also of the protein structural parameters as described below.…”

“…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 N c is the number of ionizing groups in the analyte by accounting also different positions that each one of them occupies in the macromolecular chain, both as amino acid residual and terminal amino and carboxylic groups. Thus, from one pH another, one expects to find deviations of the values taken by acid dissociation constants of ionizing groups in proteins and peptides, and hence the estimation of pK-shifts are required [15,[22][23][24][25][26][27][28][29][30]. In particular, the evaluation and prediction of pK-shifts of ionizing groups of amino acids residues in proteins have gained much attention in order to understand better complex biological systems.…”

“…The model is solved numerically by studying four proteins, for which CZEeffective mobilities and full protocol data are available in [15]. Also, emphasis is placed on the mathematical strategy to solve the resulting well-posed problem, by identifying the hydration number, estimated at a given pH, and the associated particle shape, through a consistent and convergent computational procedure presented in [15,17,22]. Then Section 3 analyses and discusses numerical results of the PLLCEM providing relevant physicochemical properties of the four proteins studied here.…”