Anal. Chem. 1998, 70, 1581-1584
Measurement of Protein Charge and Ion Binding Using Capillary Electrophoresis Manoj K. Menon and Andrew L. Zydney*
Department of Chemical Engineering, University of Delaware, Newark, Delaware 19716
A new technique is described for the rapid and accurate measurement of electrophoretic mobilities of proteins in different solution environments using capillary electrophoresis. Data were obtained at different pH using surface-modified capillaries to reduce nonspecific protein adsorption and using hydrodynamic mobilization to improve reproducibility and overall accuracy. The net protein charge and extent of anion binding were evaluated from the mobility data obtained in different pH and ionic environments for bovine serum albumin. The results were in good agreement with titration data obtained using ion-selective electrodes and mobility data obtained using free solution electrophoresis. The method requires extremely small amounts of protein (picogram quantities and nanoliter volumes) and is easily automated, making it very suitable for protein characterization and for initial screening of possible separation techniques. Electrostatic interactions can significantly affect the stability, activity, conformation, and purification/isolation of many proteins. The charge characteristics of a given protein are determined by the extent of ionization of the various acidic and basic amino acid residues in combination with the binding of specific anions, cations, and charged ligands. The magnitude of the protein charge is thus a complex function of the pH, ionic strength, and detailed composition of the electrolyte solution. Previous studies have generally evaluated protein charge and ion binding by direct titration, equilibrium dialysis, free solution electrophoresis, or NMR. Titration experiments evaluate the extent of H+ and ion binding from the change in free concentration of a given ion upon addition of a known amount of that species. Glass electrodes are typically used for pH measurement. Ionselective electrodes are required to study the binding of specific anions or cations. Equilibrium dialysis experiments are based on the Donnan re-distribution of ions across a semipermeable membrane caused by the addition of a charged protein to the solution on one side of the membrane. Both the titration and dialysis methods require relatively large amounts of protein and very accurate measurement of ion concentrations since the extent of ion binding is determined from (small) differences in concentration (either before and after addition of a known volume of solution or between solutions on the two sides of the dialysis membrane). Nuclear magnetic resonance techniques can provide detailed information on the properties of specific ion binding sites; however, these techniques are awkward and expensive to use to evaluate the overall protein charge. S0003-2700(97)00902-5 CCC: $15.00 Published on Web 03/12/1998
© 1998 American Chemical Society
Electrophoretic techniques provide a direct measure of the protein mobility, with the protein charge evaluated using theoretical models that relate the mobility to the surface potential (typically by assuming a relatively simple protein shape and uniform charge distribution). The idea of using electrophoresis to study ion binding is well-established. Many investigators have used the Tiselius moving boundary technique to measure protein electrophoretic mobility in different buffers.1-3 More recently, Douglas et al.4 developed a free-flow electrophoresis cell to measure protein mobilities. However, both of these techniques require relatively large (typically microgram to milligram) quantities of protein and use complex analytical systems to evaluate the mobilities. In addition, the boundary patterns in the Tiselius technique can be difficult to quantify. Whitesides and co-workers5,6 used a technique that they termed “affinity capillary electrophoresis” to evaluate binding constants of specific charged ligands from changes in protein mobility. However, this technique was focused on binding of a high-affinity ligand to a single specific binding site on the protein. The technique described in this paper can be used to evaluate the extent of ion binding from the background electrolyte over a wide range of solution conditions, including conditions where the protein may be positively or negatively charged. The importance of intermolecular electrostatic interactions in protein solutions provided the motivation for the development of a technique for the rapid and accurate measurement of electrophoretic mobilities, protein charge, and ion binding using available capillary electrophoresis equipment. The usefulness of this approach is demonstrated for the protein bovine serum albumin (BSA), with data obtained in different buffer solutions used to evaluate the protein charge as a function of pH as well as the extent of anion (chloride and perchlorate) binding. EXPERIMENTAL METHODS Bovine serum albumin fraction V Powder (A7906), fatty acidfree BSA (A7030), BisTris, Tris, and HCl were all obtained from Sigma Chemical (St. Louis, MO). Sodium acetate and sodium phosphate were obtained from Fisher Scientific (Pittsburgh, PA). (1) Longsworth, L. G.; Jacobsen, C. F. J. Phys. Colloid Chem. 1949, 53, 126135. (2) Schlessinger, B. S. J. Phys. Chem. 1958, 62, 916-920. (3) Norde, W.; Lyklema J. J. Colloid Interface Sci. 1978, 66, 277-284. (4) Douglas, N. G.; Humffray, A. A.; Pratt, H. R. C.; Stevens, G. W. Chem. Eng. Sci. 1995, 50, 743-754. (5) Gomez, F. A.; Avila L. Z.; Chu, Y.-H.; Whitesides, G. M. Anal. Chem. 1994, 66, 1785-1791. (6) Chu, Y.-H.; Avila L. Z.; Gao, J.; Whitesides, G. M. Acc. Chem. Res. 1995, 28, 461-468.
Analytical Chemistry, Vol. 70, No. 8, April 15, 1998 1581
Sodium perchlorate and perchloric acid were obtained from Aldrich (Milwaukee, WI), and sodium chloride was obtained from EM Sciences (Gibbstown, NJ). All chemicals were analytical reagent grade. Mesityl oxide, obtained from Sigma Chemical, was used as a neutral marker. Salt solutions were prepared using deionized water obtained from a Nanopure water purification system (Barnstead, Dubuque, IA) with resistivity greater than 18 MΩ cm. To minimize pH variations during electrophoresis, all solutions were buffered using 1 mM sodium phosphate (pH 3-3.5), 1 mM sodium acetate (pH 3.5-5.6), 1 mM BisTris (pH 5.4-8), 1 mM Tris (pH ∼9), or 0.3 mM sodium tetraborate (pH 8-11). Solution pH was adjusted using the corresponding acid and base for a given salt (i.e., HCl and NaOH were used for NaCl solutions). pH was measured using an Acumet 915 pH meter (Fisher Scientific). Solution conductivity was determined using a Horiba ES-12 conductivity meter (Kyoto, Japan). Protein samples were prepared by dissolving the protein (BSA) in the buffer solution and adjusting the pH to the same value as the buffer. A protein concentration of 2 g/L was used in all experiments reported here. The neutral marker was prepared as a 1 mM mesityl oxide solution in the desired salt and buffer. Capillary electrophoresis was performed using an Isco model 3850 capillary electropherograph equipped with a dual-polarity variable high-voltage dc supply (0-30 kV) and a variablewavelength UV-visible absorbance detector (Isco, Lincoln, NE) with detection at 214 nm. Fused-silica capillaries (i.d. ) 75 µm, o.d. ) 363 µm, total length 50 cm, distance to detector ∼30 cm) with different surface modifications were used to minimize protein adsorption to the capillary surface. A CElect Amine capillary with a positively charged polymeric coating (Supelco Inc., Bellefonte, PA) was used at low pH (pH
