Analytical Centrifugation: Equilibrium Approach

Tom Laue1

1 University of New Hampshire, Durham
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 20.3
DOI:  10.1002/0471140864.ps2003s18
Online Posting Date:  May, 2001
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The specific interaction of biological molecules with one another is fundamental to the biochemistry of all living things. Equilibrium sedimentation is a classic method of biochemistry that provides first‐principle thermodynamic information about the molar mass, association energy, association stoichiometry, and thermodynamic nonideality of molecules in solution. It is one of the most rigorous, powerful, and readily adapted methods for characterizing solution interactions.

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Table of Contents

  • Theory
  • Strategy
  • Data Analysis and Interpretation
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

   Arakawa, T. and Timasheff, S.N. 1985. Calculation of the partial specific volume of proteins in concentrated salt and amino acid solutions. Methods Enzymol. 117:60‐65.
   Casassa, E.F. and Eisenberg, H. 1964. Thermodynamic analysis of multicomponent solutions. Adv. Protein Chem. 19:287‐395.
   Christopherson, R.I., Jones, M.E., and Finch, L.R. 1979. A simple centrifuge column for desalting protein solutions. Anal. Biochem. 100:184‐187.
   Fleming, K.G., Ackerman, A.L., and Engelman, D.M. 1997. The effect of point mutations on the free energy of transmembrane alpha‐helix dimerization. J. Mol. Biol. 272:266‐275.
   Gray, R.A., Stern, A., Bewley, T., and Shire, S.J. 1995. Rapid Determination of Spectrophotometric Absorptivity by Analytical Ultracentrifugation. Application Data Sheet A‐1815A. Beckman Coulter Instruments, Fullerton, Calif.
   Haschemeyer, R.H. and Bowers, W.F. 1970. Exponential analysis of concentration or concentration difference data for discrete molecular weight distributions in sedimentation equilibrium. Biochemistry 9:435‐445.
   Johnson, M.L., Correia, J.J., Yphantis, D.A., and Halvorson, H.R. 1981. Analysis of data from the analytical ultracentrifuge by nonlinear least‐squares techniques. Biophys. J. 36:575‐588.
   Laue, T.M. 1992. Short Column Sedimentation Equilibrium Analysis for Rapid Characterization of Macromolecules in Solution. Technical Information DS835. Beckman Instruments, Palo Alto, Calif.
   Laue, T.M. 1995. Sedimentation equilibrium as a thermodynamic tool. Methods Enzymol. 259:427‐452.
   Laue, T.M. 1996. Solution Interaction Analysis: Choosing Which Optical System of the Optima XL‐I Analytical Ultracentrifuge to Use. Application data sheet A‐1821A. Beckman Coulter Instruments, Fullerton, Calif.
   Laue, T.M., and Stafford, W.F. III 1999. Modern applications of analytical ultracentrifugation. Annu. Rev. Biophys. Biomol. Struct. 28:75‐100.
   Laue, T.M., Shah, B.D., Ridgeway, T.M. and Pelletier, S.L. 1992. Computer‐aided interpretation of analytical sedimentation data for proteins. In Analytical Ultracentrifugation in Biochemistry and Polymer Science (S.E. Harding, A.J. Rowe, and J.C. Horton, eds.) pp. 90‐125. Royal Society of Chemistry, Cambridge.
   Laue, T.M., Senear, D.F., Eaton, S.F., and Ross, J.B.A. 1993. 5‐Hydroxytryptophan as a new intrinsic probe for investigating protein‐DNA interactions by analytical ultracentrifugation. Study of the effect of DNA on self‐assembly of the bacteriophage cI repressor. Biochemistry 32:2469‐2472.
   Luckow, E.A., Lyons, D.A., Ridgeway, T.M., Esmon, C.T., and Laue, T.M. 1989. Analysis of the interaction of coagulation factor V heavy chain with prothrombin and prethrombin‐1 by analytical ultracentrifugation. Role of activated protein C in regulating this interaction. Biochemistry 28:2348‐2454.
   McRorie, D.K. and Voelker, P.J. 1993. Self‐associating Systems in the Analytical Ultracentrifuge. Beckman Coulter Instruments, Fullerton, Calif.
   Morris, M. and Ralston, G.B. 1985. Determination of the parameters of self‐association by direct fitting of the omega function. Biophys. Chem. 23:49‐61.
   Ralston, G.B. 1993. Introduction to Analytical Ultracentrifugation. Beckman Coulter Instruments, Fullerton, Calif.
   Reynolds, J.A. and McCaslin, D.R. 1985. Determination of protein molecular weight in complexes with detergent without knowledge of binding. Methods Enzymol. 117:41‐53.
   Rickwood, D. 1984. Centrifugation: A Practical Approach, 2nd ed. IRL Press, Washington, D.C.
   Rivas, G.A. and Minton, A.P. 1993. New developments in the study of biomolecular associations via sedimentation equilibrium. Trends Biochem. Sci 18:284‐287.
   Roark, D.E. and Yphantis, D.A. 1969. Studies of self‐associating systems by equilibrium ultracentrifugation. Ann. N.Y. Acad. Sci 164:245‐278.
   Schuck, P. 1994. Simultaneous radial and wavelength analysis with the Optima XL‐A analytical ultracentrifuge. Progr. Colloid Polym. Sci. 94:1‐13.
   Senear, D.F. and Teller, D.C. 1981. Thermodynamics of concanavalin A dimer‐tetramer self‐association: Sedimentation equilibrium studies. Biochemistry 20:3076‐3083.
   Van Holde, K.E. 1985. Sedimentation. In Physical Biochemistry, pp. 110‐136. Prentice‐Hall, Englewood Cliffs, N.J.
   Yphantis, D.A. 1960. Rapid determination of molecular weights of peptides and proteins. Ann. N.Y. Acad. Sci. 88:586‐601.
   Yphantis, D.A. 1964. Equilibrium ultracentrifugation of dilute solutions. Biochemistry 3:297‐317.
   Yphantis, D.A., Correia, J.J., Johnson, M.L., and Wu, G. 1978. Detection of heterogeneity in self‐associating systems. In Physical Aspects of Protein Interactions (N. Catsimpoolas, ed.) pp. 275‐303. Elsevier/North‐Holland, Amsterdam.
Internet Resources
  Site for MATCH program (David Yphantis and Jeff Lary), used for monitoring minimized average differences in concentration profiles over time.
  Contact for Center to Advance Molecular Interaction Science (CAMIS), for assistance with data analysis.
  Contact for the ultracentrifuge users group at Boston Biomedical Research Institute, for assistance with data analysis.
  Site for Sednterp (J. Philo, D.B. Hayes, and T.M. Laue), a program for the interpretation of sedimentation data for proteins. The program uses databases so that it is readily customized and extended.
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