Using Protein Charge Ladders To Estimate the Effective Charges and Molecular Weights of Proteins in Solution

This paper describes the use of capillary electrophoresis (CE) and protein charge ladders to estimate values of effective charge (Z) and molecular weight of proteins under nondenaturing conditions. A panel of 14 proteins with a range of charges and shapes was modified by acetylation with acetic anhydride to yield protein charge ladders. A protein charge ladder is a family of derivatives of a protein that differ in integral units of charge, but minimally in hydrodynamic drag; this mixture of proteins appears in electrophoresis as a set of peaks with regular spacings. Analysis of the electrophoretic mobilities of the members of these charge ladders yields values of Z and electrophoretic coefficients: for a description of mobility based on the equation μ = CPZ(MW)-α, CP = 6.3 cm2 min-1 kV-1 charge-1 kD0.48, α = 0.48; for μ = CrZ[r(1 + κr)]-1, Cr = 55 cm2 min-1 kV-1 charge-1 Å (r is the spherical radius of the protein and κ is a function of ionic strength). The primary usefulness of charge ladders is in measuring the effective charge, Z, of proteins in solution; this information is difficult to obtain by any other procedure. A secondary value of the method is to estimate values of molecular weight. Although less general and convenient than SDS−PAGE, this method allows estimates of molecular weight of nondenatured proteins and is thus applicable to oligomers, noncovalent aggregates, proteins with multiple, non-cross-linked chains, and other systems to which SDS−PAGE is not applicable. The values of molecular weight calculated using the electrophoretic mobilities of proteins in solution and the above constants agreed with literature values to within 20% (with an ambiguous result for ovalbumin). A combination of this technique and SDS−PAGE will be useful in estimating the number of subunits or stage of aggregation of proteins in solution.