Determination of the effective charge of a protein in solution by capillary electrophoresis.

Abstract

This paper describes two methods to estimate the effective charge of a protein in solution by capillary electrophoresis and demonstrates these methods by using representative proteins. In one method, a "charge ladder"-a series of derivatives of a protein differing by known increments of charge but differing only minimally in hydrodynamic drag-is generated by covalent modification of the e-amino groups of lysines with 4-sulfophenyl isothiocyanate or acetic anhydride. In the second method, the equivalent of a charge ladder is produced by noncovalent association of a protein with differently charged ligands. (1) is straightforward and can be used to estimate the net charge of a protein over a range of values of pH. This method, however, requires substantial quantities (2-10 mg) of pure protein, and the measurement may be influenced by association of the protein with ions in solution. Winzor and Ojteg (2, 3) have estimated net charges of proteins based on Donnan potential measurements. These methods permit the charge ofa protein to be determined over a range ofionic composition and values ofpH but also require significant quantities (1-10 mg) of a protein.Here we report two related procedures for estimating the effective charge of a protein in solution at arbitrary values of pH by using capillary electrophoresis (CE) (4-8). The two procedures are complementary, but both rely on the same strategy: to generate and compare the electrophoretic mobilities of a protein with a series of derivatives of the protein that differ in effective charge by simple multiples of a unit charge but that differ only minimally in hydrodynamic drag.

Electrophoresis of Protein Charge Ladders: A Comparison of Experiment with Various Continuum Primitive Models

“…The combination of CE and charge ladders has been used for the characterization of a variety of physical properties and interactions of proteins: examples include their net charges, 1 hydrodynamic sizes, 2 molecular weights, 3 dissociation of titratable groups, 4,5 and stabilities. 6 Charge ladders are produced by the partial and chemical modification of charged groups on the protein.…”

“…7 In free solution capillary electrophoresis, these modified proteins separate into distinct bands, or "rungs", in which each rung consists of species with the same number of chemical modifications due to similarities in their net charges. [1][2][3][4][5][6][7] As investigators have attempted to extract more quantitative information from these charge ladder studies, such as the average charge difference, ∆Z, between successive rungs of a ladder, it has become apparent that more realistic modeling of the free solution electrophoresis of proteins is needed. Up to the present, much of the analysis has been based on the Henry model, 8 in which the protein is modeled as a solid sphere with a centrosymmetric charge distribution.…”

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“…Charges on the protein can affect substrate binding and reactivity [13], protein-protein interactions [14] and secretion [15], localization of specific protein. Besides, osmotic pressure of a cell is affected by the sum of the charges of all of its protein [12]. In proteins arginine and lysine are the two residues predominantly glycated.…”

“…Overall hyperglycemia and AGEs directly or indirectly is a major factor in the pathogenesis of diabetes, neurodegenerative diseases, as well as in the process of aging [9][10][11] Arginine and lysine being positively charged contributes significantly to the net charge of the protein. The net charge on the protein is the summation of charges carried by its electrostatic components including, charged amino acid residues, metal ions and charged cofactors [12]. However the effective charge of a protein is always less than the net charge and is determined by the extent of screening of the charge by the association of the protein by counter ions in the surrounding medium [12].…”

“…The net charge on the protein is the summation of charges carried by its electrostatic components including, charged amino acid residues, metal ions and charged cofactors [12]. However the effective charge of a protein is always less than the net charge and is determined by the extent of screening of the charge by the association of the protein by counter ions in the surrounding medium [12]. Charges on the protein can affect substrate binding and reactivity [13], protein-protein interactions [14] and secretion [15], localization of specific protein.…”