RULES RELATING ELECTROPHORETIC MOBILITY, CHARGE AND MOLECULAR-SIZE OFPEPTIDES AND PROTEINS

The absence of supporting media in free solution high-performance capi llary electrophoresis (HPCE) makes it an ideal system for the study of the relationship between electrophoretic mobility (mu(em)) and the mo lecular size and charge of proteins and peptides. In this review, the theory of electrophoresis, developed for rigid, insulating, spherical particles, is modified to develop models for the electrophoretic behav iour of proteins and peptides. For a given set of experimental conditi ons, mu(em) of a protein/peptide is proportional to its charge (q) and is inversely proportional to its Stoke's radius (r). Furthermore, mu( em) is most sensitive to changes in q and, as a consequence, the relia bility of equations relating mu(em) to protein/peptide q and r is depe ndent upon the accurate calculation or determination of q. For conveni ence, q and r of proteins and peptides are generally expressed in term s of calculated valence (Z(c)) and molecular mass (M), respectively, b oth of which can be determined from the amino acid sequence of the pro tein/peptide. However, the calculation of q using Z(c) is made more co mplex by the effects of electrostatic charge suppression, such that Z( c) is an overestimation of actual charge. Charge suppression becomes i ncreasingly significant as the protein/peptide charge increases, such that, for peptides, the relationship between q and Z(c) can be approxi mated by a logarithmic function. The mu(em) for peptides, therefore, c an be approximated by the equation: mu(em) = ln(Z(c) + 1)/K M-s where s varies between 1/3 and 2/3, and K is a constant that is valid for a particular set of experimental conditions. The rather simplistic compe nsation for charge suppression in this equation is inadequate for prot eins where the magnitude of charge suppression is greater and the mech anisms are more complex. For proteins, the relationship suggested for the prediction of mu(em) from Z(c) and M is: mu(em) = Z(c)/KFzMs where s again varies between 1/3 and 2/3 and F-z is a pH-independent propor tionality factor defined as the quotient, Z(c)/Z(a), with Z(a) being a ctual protein valence. The factor F-z can be determined empirically, h owever, it is valid only for the particular set of experimental condit ions under which it is determined. For peptides, the mass exponent, s, approaches 1/3 when the peptides have high charge densities and open structures. However, s approaches 1/2 for peptides with lower charge d ensities that are capable of more randomized motion during electrophor esis. Finally, s approaches 2/3 for proteins, suggesting that the fric tional forces acting on a protein undergoing electrophoretic motion ar e proportional to the surface area of these larger, more rigid, struct ures. In conclusion, the development of relationships between mu(em), M and Z(c) for peptides and proteins offers a powerful tool, not only for predicting electrophoretic mobility, but also for optimising HPCE separations, studying structural modifications (e.g. phosphorylation, glycosylation, deamidation, etc.), and for the investigation of surfac e charge characteristics and conformation. (C) 1997 Elsevier Science B .V.