Analytical Ultracentrifugation: Sedimentation Velocity Analysis

Walter Stafford1

1 Massachusetts General Hospital, Harvard Medical School, Boston
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 20.7
DOI:  10.1002/0471140864.ps2007s31
Online Posting Date:  May, 2003
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Abstract

The analytical ultracentrifuge is a high speed centrifuge with an optical system allowing observation of the concentration of macromolecules as a function of radius and time. In sedimentation velocity experiments,relatively high speeds are used so that a boundary is formed between the solution of sedimenting macromolecule and the buffer in which it is dissolved. Analysis of the rate boundary movement and evolution of its shape can yield information about the molar masses of species present as well as stoichiometries and equilibrium constants for their interactions. This overview discusses crucial issues pertaining to sedimentation velocity experiments, including non‐interacting and interacting systems, ideality and non‐ideality, and reversible versus kinetically limited equilibrium scenarios.

     
 
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Table of Contents

  • Sedimentation Velocity Theory
  • Experimental Design and Protocols
  • Data Analysis and Interpretation
  • Tables
     
 
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Materials

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Figures

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Literature Cited

Literature Cited
   Behlke, J. and Ristau, O. 2002. A new approximate whole boundary solution of the Lamm differential equation for the analysis of sedimentation velocity experiments. Biophys. Chem. 5:59‐68.
   Cann, J.R. and Goad, W.B. 1968. Theory of transport of interacting systems of biological macromolecules. Adv. Enzymol. Relat. Areas Mol. Biol. 30:139‐177.
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   Christopherson, R.I., Jones, M.E., and Finch, L.R. 1979. A simple centrifuge column for desalting protein solutions. Anal. Biochem. 100:184‐187.
   Claesson, S. and Moring‐Claesson, I. 1961. Determination of the size and shape of protein molecules: Ultracentrifugation. In Laboratory Manual of Analytical Methods of Protein Chemistry (Including Polypeptides). (Alexander, P., ed.) Pergamon Press, New York.
   Demeler, B. and Saber, H. 1998. Determination of molecular parameters by fitting sedimentation data to finite‐element solutions of the Lamm equation. Biophys. J. 74:444‐454.
   Holladay, L. 1979. An approximate solution to the Lamm equation. Biophys. Chem. 10:187‐190.
   Holladay, L. 1980. Simultaneous rapid estimation of sedimentation coefficient and molecular weight. Biophys. Chem. 11:303‐308.
   Lamm, O. 1929. Die Differentialgleichung der Ultrazentrifugierung. Arkiv. Math. Astron. Fysik 21B(no. 2):1‐4.
   Lobert, S., Frankfurter, A., and Correia, J.J. 1995. Binding of vinblastine to phosphocellulose‐purified and alpha beta‐class III tubulin: The role of nucleotides and beta‐tubulin isotypes. Biochemistry 34:8050‐8060.
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   Na, G.C. and Timasheff, S.N. 1986a. Interaction of vinblastine with calf brain tubulin: Multiple equilibria. Biochemistry 25:6214‐6222.
   Na, G.C. and Timasheff, S.N. 1986b. Interaction of vinblastine with calf brain tubulin: Effects of magnesium ions. Biochemistry 25:6222‐6228.
   Philo, J.S. 1997. An improved function for fitting sedimentation velocity data for low‐molecular‐weight solutes. Biophys. J. 72:435‐444.
   Philo, J.S. 2000. A method for directly fitting the time derivative of sedimentation velocity data and an alternative algorithm for calculating sedimentation coefficient distribution functions. Anal. Biochem. 279:151‐163.
   Rivas, G., Fernandez, J.A., and Minton, A.P. 1999. Direct observation of the self‐association of dilute proteins in the presence of inert macromolecules at high concentration via tracer sedimentation equilibrium: Theory,experiment, and biological significance. Biochemistry 38:9379‐9388.
   Rivas, G., Stafford, W.F., and Minton, A.P. 1999. Characterization of heterologous protein‐protein interaction via analytical ultracentrifugation. Methods 19:194‐212.
   Schachman, H.K. 1959. Ultracentrifugation in Biochemistry. Academic Press, New York.
   Schuck, P. 1998. Sedimentation analysis of noninteracting and self‐associating solutes using numerical solutions to the Lamm equation. Biophys. J. 75:1503‐1512.
   Schuck, P. 2000. Size‐distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78:1606‐1619.
   Solovyova, A., Schuck, P., Costenaro, L., and Ebel, C. 2001. Non‐ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents. Biophys. J. 81:1868‐1880.
   Stafford, W.F. 1992. Boundary analysis in sedimentation transport experiments: A procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal. Biochem. 203:295‐301.
   Stafford, W.F. 1994a. Boundary analysis in sedimentation velocity experiments. In Methods in Enzymology, Numerical Computer Methods, 240, part B (M.L. Johnson and L. Brandeds.) pp. 478‐501. Academic Press Orlando, Fla.
   Stafford, W.F. 1994b. Sedimentation boundary analysis of interacting systems: Use of the apparent sedimentation coefficient distribution function. In Modern Analytical Ultracentrifugation: Acquisition and Interpretation of Data for Biological and Synthetic Polymer Systems (T.M. Schuster and T.M. Laueeds.) pp. 119‐137. Birkhäuser Boston Boston,Mass.
   Stafford, W.F. 1998. Time difference sedimentation velocity analysis of rapidly reversible interacting systems: Determination of equilibrium constants by non‐linear curve fitting procedures. Biophys. J. 74:A301.
   Stafford, W.F. and Schuster, T.M. 1995. Hydrodynamic methods. In Introduction to Biophysical Methods for Protein and Nucleic Acid Research (J.A. Glasel and M.P. Deutschereds.) pp. 111‐145. Academic Press, San Diego.
   Stafford, W.F. and Sherwood, P. 2003. Analysis of heterologous interacting systems by sedimentation velocity: Curve‐fitting algorithms for estimation of sedimentation coefficients and equilibrium constants. J. Biophys. Chem. In press
   Tanford, C. 1961. Physical Chemistry of Macromolecules. John Wiley & Sons, New York.
   Timasheff, S.N., Frigon, R.P., and Lee, J.C. 1976. A solution physical‐chemical examination of the self‐association of tubulin. Fed. Proc. 35:1886‐1891.
   Todd, G.P. and Haschmeyer, R.H. 1981. General solution to the inverse problem of the differential equation of the ultracentrifuge. Proc. Natl. Acad. Sci. U.S.A. 78:6739‐6743.
   Van Holde, K.E. and Weischet, W.O. 1978. Boundary analysis of sedimentation‐velocity experiments with monodisperse and paucidisperse solutes. Biopolymers 17:1387‐1403.
   Waugh, D.F. and Yphantis, D.A. 1952. Rotor temperature measurement and control in the ultracentrifuge. Rev. Sci. Inst. 23:609‐614.
   Williams, J.W., Van Holde, K.E., Baldwin, R.L., and Fujita, H. 1958. The theory of sedimentation analysis. Chem. Rev. 58:715‐806.
Key References
   Williams et al., 1958. See above.
  This selection of three pieces of older literature contains a tremendous amount of information that is very germane to modern day sedimentation analysis.
   Schachman, 1959. See above.
   Claesson and Moring‐Claesson, 1961. See above.
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