Studies of Macromolecular Interaction by Sedimentation Velocity

  • James C. Lee
  • Surendran Rajendran
Part of the Emerging Biochemical and Biophysical Techniques book series (EBBT)


As our knowledge expands and new systems are investigated, it becomes clear that elucidating precise biochemical regulatory mechanisms requires detailed understanding of macromolecular assembly processes. For example, the regulation of gene expression involves an intricate network of protein-protein and protein-nucleic acid interactions and the mechanism of some allosteric enzymes is linked to subunit assembly. In order to define these mechanisms one needs information on the identities of proteins in the complex, the affinities of these proteins for each other and the effects of regulators on the formation of these complexes. A direct way of studying macromolecular assembly is to monitor the resulting changes in mass as a consequence of the formation of these macromolecular complexes. One of the methods that enables one to directly monitor the mass of macromolecules is the transport technique. Among the transport methods sedimentation analysis is the technique of choice because of the sound fundamental principles on which the method is based and because of its resolving power. Excellent reviews on the applications of sedimentation equilibrium in studying macromolecular self-associations and heteropolymers formation are included in Part I of this volume. In this chapter the focus is on applications of sedimentation velocity. One of the advantages of sedimentation velocity over that of equilibrium is its speed of analysis, e.g., a run can be completed within an hour whereas equilibrium experiments may take much longer. Hence, if a biological sample is unstable the study on that system may have to be conducted using sedimentation velocity. However, a larger amount of sample will be required for velocity analysis than for equilibrium measurements, e.g., the calf brain tubulin and rabbit muscle phosphofructokinase systems require a few mg of protein to define a curve of sedimentation coefficient vs concentration.


Sedimentation Velocity Infinite Dilution Sedimentation Pattern Sedimentation Coefficient High Protein Concentration 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Basch, J.J. and Timasheff, S.N. (1967): Hydrogen ion equilibria of the genetic variants of bovine ß-lactoglobulin. Arch. Biochem. Biophysics 118: 37–47.CrossRefGoogle Scholar
  2. Cann, J.R. (1970): Interacting Macromolecules — The Theory and Practice of Their Electrophoresis, Ultracentrifugation, and Chromatography. 93–151 New York: Academic Press.Google Scholar
  3. Cann, J.R. and Goad, W.B. (1970): Bimodal sedimenting zones due to ligand-mediated interactions. Science 170: 441–445.PubMedCrossRefGoogle Scholar
  4. Cann, J.R. and Goad, W.B. (1972): Theory of sedimentation for ligand- mediated dimerization. Arch. Biochem. Biophys. 153: 603–609.PubMedCrossRefGoogle Scholar
  5. Cann, J.R. (1973): Theory of zone sedimentation for non-cooperative ligand- mediated interactions. Biophys. Chem. 1: 1–10.CrossRefGoogle Scholar
  6. Cann, J.R. and Goad, W.B. (1973): Measurements of protein interactions medicated by small molecules using sedimentation velocity. Methods in Enzymology 27: 296–306.PubMedCrossRefGoogle Scholar
  7. Cox, D.J. (1978): Calculation of simulated velocity profiles for self- associating solutes. Methods in Enzymology 48: 212–242.CrossRefGoogle Scholar
  8. Cox, D.J. and Dale, R.S. (1981): Simulations of transport experiments for interacting systems. In: Protein-Protein Interactions 173–211, New York: Wiley Interscience Publications.Google Scholar
  9. Frigon, R.P. and Timasheff, S.N. (1975a): Magnesium-induced self- association of calf brain tubulin. I. Stoichiometry. Biochemistry 14: 4559–4566.PubMedCrossRefGoogle Scholar
  10. Frigon, R.P. and Timasheff, S.N. (1975b): Magnesium-induced self- association of calf brain tubulin. II. Thermodynamics. Biochemistry 14: 4567–4572.PubMedCrossRefGoogle Scholar
  11. Fujita, H. (1962): Mathematical theory of Sedimentation Analysis. New York: Academic Press.Google Scholar
  12. Gilbert, G.A. (1955): Discuss. Faraday Soc. 20: 68–71.Google Scholar
  13. Gilbert, L.M. and Gilbert, A.G. (1973): Sedimentation velocity measurement of protein association. Methods in Enzymology 27: 273–296.PubMedCrossRefGoogle Scholar
  14. Hesterberg, L.K. and Lee, J.C. (1981): Self-association of rabbit muscle phosphofructokinase at pH 7.0: Stoichiometry. Biochemistry 20: 2974–2980.PubMedCrossRefGoogle Scholar
  15. Holloway, R.R. and Cox, D.J. (1974): Computer simulation of sedimentation in the ultracentrifuge. VII. Solutes undergoing indefinite self-association. Arch. Biochem. Biophys. 160: 595–602.PubMedCrossRefGoogle Scholar
  16. Johnson, P. and Shooter, E.M. (1950): The globulin of the groundnut. I. Investigation of arachin as a dissociation system. Biochem. Biophys. Acta 5: 361.PubMedCrossRefGoogle Scholar
  17. Kirkwood, J.G. (1954): The general theory of irreversible processes in solutions of macromolecules. J. Polym. Sci. 12: 1–14.CrossRefGoogle Scholar
  18. Kumosinski, T.F. and Timasheff, S.N. (1966): Molecular interactions in B- lactoglobulin. X. The stoichiometry of the B-lactoglobulin mixed tetramerization. J. Am. Chem. Soc. 88: 5635–5642.CrossRefGoogle Scholar
  19. Lee. J.C., Harrison, D. and Timasheff, S.N. (1975): Interaction of vinblastine with calf brain microtubule protein. J. Biol. Chem. 250: 9276–9282.PubMedGoogle Scholar
  20. Na, G.C. and Timasheff, S.N. (1980): Stoichiometry of the vinblastine - induced self-association of calf brain tubulin. Biochemistry 19: 1347–1354.PubMedCrossRefGoogle Scholar
  21. Nichol, L.W., Bethune, J.L., Kegeles, G. and Hess, E.L. (1964): Interacting Protein Systems. In: The Proteins, H. Neurath, ed., Vol II 2nd Ed. 305 - 403. New York: Academic Press.Google Scholar
  22. Schachman, H.K. (1959): Ultracentrifugation in Biochemistry. New York, Academic Press.Google Scholar
  23. Timasheff, S.N. and Townend, R. (1960): Molecular interactions in 15- lactoglobulin. I. The electrophoretic heterogeneity of B- lactoglobulin close to its isoelectric point. J. Am. Chem. Soc. 82: 3157–3161.CrossRefGoogle Scholar
  24. Timasheff, S.N. and Townend, R. (1961): Molecular interactions in B- lactoglobulin. V. The association of the genetic species of B- lactoglobulin below the isoelectric point. J. Am. Chem. Soc. 83: 464–469.CrossRefGoogle Scholar
  25. Timasheff, S.N., Andreu, J.M. and Na, G.C. (1991): Physical and spectroscopic methods for the evaluation of the interactions of antimitotic agents with tubulin. Pharmac. Ther. 52: 191–210.CrossRefGoogle Scholar
  26. Townend, R., Winterbottom, R.J. and Timasheff, S.N. (1960a): Molecular interactions in B-lactoglobulin. II. Ultracentrifugai and electrophoretic studies of the association of B-lactoglobulin below its isoelectric point. J. Am. Chem. Soc. 82: 3161–3168.CrossRefGoogle Scholar
  27. Townend, R. and Timasheff, S.N. (1960): Molecular interactions in B- lactoglobulin. III. Light scattering investigation of the stoichiometry of the association between pH 3.7 and 5.2. J. Am. Chem. Soc. 82: 3168–3174.CrossRefGoogle Scholar
  28. Townend, R., Weinberger, L. and Timasheff, S.N. (1960b): Molecular interactions in B-lactoglobulin. IV. The dissociation of B- lactoglobulin below pH 3.5. J. Am. Chem. Soc. 82: 3175–3179.CrossRefGoogle Scholar
  29. Yphantis, D.A. and Waugh, D.F. (1956): Ultracentrifugai characterization by direct measurement of activity. II. Experimental. J. Phys. Chem. 60: 630–635.CrossRefGoogle Scholar

Copyright information

© Birkhäuser Boston 1994

Authors and Affiliations

  • James C. Lee
  • Surendran Rajendran

There are no affiliations available

Personalised recommendations