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.
Capillary electrophoresis (CE) is an analytical method that is useful for investigating processes that modify the charge of proteins. This paper explores the ability of CE to rationalize charges and electrophoretic mobilities of a simple protein--insulin and its acylated derivatives--as a function of pH. Insulin is a peptide hormone (MW = 5700) that has two alpha-amino groups (G alpha and F alpha) and one epsilon-amino group (K epsilon). Treatment of insulin with acetic anhydride affords seven derivatives that differ in the sites of acetylation of the three amino groups. Analysis of the pH dependence of the electrophoretic mobilities of these derivatives gives pKa values for the two N-terminal ammonium groups: pKa (G alpha) = 8.4; pKa (F alpha) = 7.1. Values of the total charge of insulin estimated from electrophoretic mobility differ from those estimated from values of pKa for its ionizable groups by less than 0.5 unit for both bovine and human insulins over the range of pH from 5.5 to 9.5. Analysis of the concentration dependence of the electrophoretic mobility of insulin yields a lower limit for the association constant for dimerization of insulin of KD > or = 6 x 10(3) M-1 (25 mM tris and 192 mM Gly, pH 8.4). Studies of electrophoretic mobility as a function of pH and extent of acetylation of amino groups rationalize the charge of insulin in detail. The sensitivity of CE to charge permits the quantitative study of electrostatic properties of proteins in solution. Insulin is a useful small-protein model with which to investigate phenomena in electrophoresis.
This paper describes the estimation of binding constants (Kb) between carbonic anhydrase B (CAB, EC 4.2.1.1, from bovine erythrocytes) and charged benzenesulfonamides by affinity capillary electrophoresis (ACE) under conditions in which the migration time is affected by changes in electroosmotic flow and by nonspecific interactions accompanying changes in the concentration of ligand. Comparisons of values of migration times of the protein of interest, and of "noninteracting" marker proteins, with those of a neutral internal standard provide the basis for corrections for variable electroosmotic flow; these corrections make possible the estimation of Kb and its uncertainty even in the presence of substantial variations in electroosmotic flow.
A procedure is reported for the conversion of tert-butyldimethylsilyl chloride and cioso-l,2-C2B10H12 (1) to the corresponding silylcarborane derivative 2 in 99% yield. Compound 2 served as a valuable synthon to other monosubstituted carboranyl derivatives. Lithiation and reaction at the carborane 2-vertex with a variety of monoand difunctional electrophiles under mild conditions formed the corresponding silylated products. Subsequent deprotection with tetrabutylammonium fluoride produced the corresponding monosubstituted carborane derivatives in high yield.
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