Protein Charge Determination
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- Abstract
- Table of Contents
- Figures
- Literature Cited
Abstract
The most popular current method of determining protein valence entails the calculation of net charge from amino acid sequence/composition. However, the inaccuracy of that approach was recognized long before the advent of the protein data banks and computer programs to facilitate its adoption. Capillary zone electrophoresis affords the simplest and most economical procedure for obtaining a reliable estimate of the net charge of a protein in the buffer system of interest. This unit explains the major pitfalls in the calculation of net charge from protein sequence data.
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- Calculation of Protein Charge from Sequence Data
- Electrophoretic Determination of Net Protein Charge
- Relationship Between Mobility and Protein Charge
- Valence Measurement by the “Charge Ladder” Approach
- Concluding Remarks
- Literature Cited
- Figures
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Figure 2.10.1 Effect of ionic strength ( I ) on the isoelectric point of aldolase in acetate and phosphate buffers. The data, taken from Velick (), were acquired by interpolating electrophoretic mobility measurements obtained by the moving boundary method. View Image -
Figure 2.10.2 Time course of absorbance (200 nm) at the detection point, located at distance x (20 cm) from the point of sample application, in a zonal capillary electrophoresis experiment on β‐lactoglobulin in 0.05 M phosphate buffer, pH 6.7. t P , the time required for the protein zone to migrate the 20 cm under the influence of an electric field strength ( E ) of 260 V/cm, is the protein characteristic required for determination of electrophoretic mobility ( u P ) from Equation . The electrophoretic time‐course has been inferred from Figure b in Gao et al. (). View Image -
Figure 2.10.3 pH dependence of the net charge for ovalbumin ( I = 0.1 M) deduced from published electrophoretic mobility measurements (Longsworth, ; Creeth and Winzor, ). Open symbols denote values calculated by the application of Equation , which assumes spherical geometry for the protein, whereas closed symbols are values of z P based on a modified version of Equation that takes into account the prolate‐ellipsoidal shape of ovalbumin. View Image -
Figure 2.10.4 Estimation of protein valence by capillary zone electrophoresis and the “charge ladder” approach. Published electrophoretic mobilities for Staphylococcal nuclease and mutant variants (Table 4 of Kálmán et al., ) are plotted in accordance with Equation , the quantitative basis for valence estimation by the “charge ladder” method. The designated value of z P for each set of conditions is the reciprocal of the slope of the best‐fit description of the data in such terms. View Image
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Literature Cited
| Literature Cited | |
| Abramson, H.A., Moyer, L.S., and Gorin, M.H. 1942. Electrophoresis of Proteins. Reinhold, New York. | |
| Barnett, L.B. and Bull, H.B. 1960. Electrophoresis of ribonuclease and of β‐lactoglobulin: Isoelectric points of proteins. Arch. Biochem. Biophys. 89:162‐172. | |
| Butterman, M., Tietz, D., Orbán, L., and Chrambach, A. 1988. Ferguson plots based on absolute mobilities in polyacrylamide gel electrophoresis: Dependence of linearity of polymerization conditions and application to the determination of free mobility. Electrophoresis 9:293‐298. | |
| Carr, C.W. 1953. Studies on the binding of small ions in protein solutions with the use of membrane electrodes. III. The binding of chloride in solutions of various proteins. Arch. Biochem. Biophys. 46:417‐423. | |
| Carr, C.W. 1956. Studies on the binding of small ions in protein solutions with the use of membrane electrodes. VI. The binding of sodium and potassium ions in solutions of various proteins. Arch. Biochem. Biophys. 62:476‐484. | |
| Compton, B.J. and O'Grady, E.A. 1991. Role of charge suppression and ionic strength in free zone electrophoresis of proteins. Anal. Chem. 63:2597‐2602. | |
| Creeth, J.M. and Winzor, D.J. 1962. Physicochemical studies on ovalbumin 4. Characterization of an iodine‐modified derivative by electrophoresis and sedimentation. Biochem. J. 83:566‐574. | |
| Debye, P. and Hückel, E. 1923. Theory of electrolytes. I. Lowering of freezing point and related phenomena. Physik. Z. 24:185‐206. | |
| Ferguson, K.A. 1964. Starch gel electrophoresis: Application to the classification of pituitary proteins and polypeptides. Metabolism 13:985‐1002. | |
| Gao, J., Gomez, F.A., Härter, R., and Whitesides, G.M. 1994. Determination of the effective charge of a protein in solution by capillary zone electrophoresis. Proc. Natl. Acad. Sci. U.S.A. 91:12027‐12030. | |
| Gao, J.Y., Dubin, P.L., and Muhoberac, B.M. 1997. Measurement of the binding of proteins to polyelectrolytes by frontal analysis continuous capillary electrophoresis. Anal. Chem. 69:2945‐2951. | |
| Harding, S.E., Horton, J.C., Jones, S., Thornton, J.M., and Winzor, D.J. 1999. COVOL: An interactive program for evaluating second virial coefficients from the triaxial shape or dimensions of rigid macromolecules. Biophys. J. 76:2432‐2438. | |
| Hedrich, J.L. and Smith, A.J. 1968. Size and charge isomer separation and estimation of molecular weights by disk electrophoresis. Arch. Biochem. Biophys. 126:155‐164. | |
| Henry, D.C. 1931. The cataphoresis of suspended particles. I. The equation of cataphoresis. Proc. R. Soc. London A. 133:106‐129. | |
| Hjertén, S. 1985. High‐performance electrophoresis: Elimination of electroendosmosis and solute sorption. J. Chromatogr. 347:191‐198. | |
| Jakubke, H. and Jeschkeit, H. 1977. Amino Acids, Peptides and Proteins. John Wiley & Sons, New York. | |
| Jeffrey, P.D., Nichol, L.W., Turner, D.R., and Winzor, D.J. 1977. The combination of molecular covolume and frictional coefficient to determine the shape and axial ratio of a rigid macromolecule. J. Phys. Chem. 81:566‐574. | |
| Kálmán, F., Ma, S., Fox, R.O., and Horváth, C. 1995. Capillary electrophoresis of S. nuclease mutants. J. Chromatogr. A 705:135‐154. | |
| Linderstrøm‐Lang, K. 1924. The ionization of proteins. Compt. Rend. Trav. Lab. Carlsberg 15(7):1‐29. | |
| Linderstrøm‐Lang, K. and Nielsen, S.O. 1959. Acid−base equilibria in proteins. In Electrophoresis: Theory, Methods and Applications (M. Bier, ed.) pp. 35‐89. Academic Press, New York. | |
| Longsworth, L.G. 1941. The influence of pH on the mobility and diffusion of ovalbumin. Ann. N.Y. Acad. Sci. 41:267‐285. | |
| Overbeek, J.T.G. and Lijklema, J. 1959. Electric potentials in colloidal systems. In Electrophoresis: Theory, Methods and Applications (M. Bier, ed.) pp. 1‐33. Academic Press, New York. | |
| Rodbard, D. and Chrambach, A. 1971. Estimation of the molecular radius, free mobility and valence using polyacrylamide gel electrophoresis. Anal. Biochem. 40:95‐134. | |
| Scatchard, G., Coleman, J.S., and Shan, A.L. 1957. Physical chemistry of protein solutions. VII. The binding of some small ions to bovine serum albumin. J. Am. Chem. Soc. 79:12‐20. | |
| Skoog, B. and Wichman, A. 1986. Calculation of the isoelectric points of polypeptides from the amino acid composition. Trends Anal. Chem. 5:82‐83. | |
| Tanford, C., Hauenstein, J.D., and Rands, D.G. 1955a. Phenolic hydroxyl ionization in proteins. II. Ribonuclease. J. Am. Chem. Soc. 77:6409‐6413. | |
| Tanford, C., Swanson, S.A., and Shore, J.S. 1955b. Hydrogen ion equilibria of bovine serum albumin. J. Am. Chem. Soc. 77:6414‐6428. | |
| Tiselius, A. 1937. A new apparatus for electrophoretic analysis of colloidal mixtures. Trans. Faraday Soc. 33:524‐531. | |
| Velick, S.F. 1949. The interaction of enzymes with small ions. I. An electrophoretic and equilibrium analysis of aldolase in phosphate and acetate buffers. J. Phys. Colloid Chem. 53:135‐149. | |
| Winzor, D.J. 2003. Classical approach to interpretation of the charge‐dependence of peptide mobilities obtained by capillary zone electrophoresis. J. Chromatogr. A1015:199‐204. | |
| Winzor, D.J. 2004. Determination of the net charge (valence) of a protein: A fundamental but elusive parameter. Anal. Biochem. 325:1‐20. | |
| Winzor, D.J., Jones, S., and Harding, S.E. 2004. Determination of protein charge by capillary electrophoresis. Anal. Biochem. 333:225‐229. |
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