Determination of the effective charge of a protein in solution by capillary electrophoresis.
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 epsilon-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. Analysis of the electrophoretic mobilities of the protein and its derivatives as a function of added charge allows the effective charge to be estimated for the unmodified protein. This type of analysis permits estimation of the effective charge of a protein without knowing its composition, structure, or amino acid sequence.
Gao
J
J
Department of Chemistry, Harvard University, Cambridge, MA 02138.
Gomez
F A
FA
Härter
R
R
Whitesides
G M
GM
D000089:Acetic Anhydrides; D000818:Animals; D001557:Benzenesulfonates; D002197:Capillary Action; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002417:Cattle; D004586:Electrophoresis / Q000379:methods; D006736:Horses; D006801:Humans; D007328:Insulin / Q000737:chemistry; D017879:Isothiocyanates; D007768:Lactalbumin / Q000737:chemistry; D008024:Ligands; D008239:Lysine; D009211:Myoglobin / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D012996:Solutions; D013552:Swine; D014357:Trypsin / Q000737:chemistry
D000089:Acetic Anhydrides; D001557:Benzenesulfonates; D007328:Insulin; D017879:Isothiocyanates; D008024:Ligands; D009211:Myoglobin; D011506:Proteins; D012996:Solutions; C031800:acetic anhydride; C015308:4-isothiocyanatobenzenesulfonate; D007768:Lactalbumin; D014357:Trypsin; D002256:Carbonic Anhydrases; D008239:Lysine