title: Using protein charge ladders to estimate the effective charges and molecular weights of proteins in solution.
abstract: 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 mu = CPZ(MW)-alpha, CP = 6.3 cm2 min-1 kV-1 charge-1 kD0.48, alpha = 0.48; for mu = C(r)Z[r(1 + kappa r)]-1, C(r) = 55 cm2 min-1 kV-1 charge-1 A (r is the spherical radius of the protein and kappa 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.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
authors.forename: J
authors.initials: J
authors.lastname: Gao
authors.forename: G M
authors.initials: GM
authors.lastname: Whitesides
mesh_terms: D000089:Acetic Anhydrides; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012996:Solutions
chemical_list: D000089:Acetic Anhydrides; D011506:Proteins; D012996:Solutions; C031800:acetic anhydride