Sedimentation

Abstract

Sedimentation is a classic method of biochemistry that provides first‐principle hydrodynamic and thermodynamic information about the purity, size, shape, molar mass, association energy, association stoichiometry and thermodynamic nonideality of molecules in solution. The fundamental measurement in sedimentation is the concentration as a function of radial position. Any of the three optical systems provides the necessary concentration profiles, making sedimentation a versatile tool for analysing biological solutions. There are two distinct sedimentation methods: sedimentation velocity and sedimentation equilibrium. Data analysis from either method uses computer programs developed around fundamental equations. Velocity sedimentation is used to check for impurities and also to characterise molecular interactions. Equilibrium sedimentation is not used as widely, but it is used to provide first‐principle insights into intermolecular interactions such as macromolecular binding and thermodynamic nonideality.

Key concepts:

  • Sedimentation velocity provides information about the size and shape of molecules in solution.

  • Sedimentation equilibrium provides information about the interactions of macromolecules in solution.

  • The quaternary structure of a molecule may be determined under different solvent conditions.

  • Mass‐action equilibria may be characterised with respect to the stoichiometry and association energies.

  • Sedimentation velocity analysis is used to detect aggregates and fragments of highly purified molecules.

  • Thermodynamic nonideality may be characterised from sedimentation equilibrium analysis.

  • Analysis programs for sedimentation velocity data often use solutions of the Lamm equation.

  • Analysis programs for sedimentation equilibrium data are based on thermodynamic first principles.

Keywords: sedimentation; diffusion; thermodynamics; hydrodynamics; molecular weight

Figure 1.

A sedimentation velocity experiment for a protein with a molecular weight of 45 000, spun at 50 000 rpm and 20°C. Concentration profiles were acquired using the refractometric optical system at 8‐min intervals 2.5 h after the start of the experiment. The boundary initially was at the meniscus radius, rm, and was moving from left to right. The rate of boundary movement yields s, while the rate of boundary spreading yields D. The sample chamber was narrower at the top than at the bottom, leading to the decrease in the plateau concentration, Cp, during sedimentation.

Figure 2.

Cutaway drawing of a Beckman Coulter XLI analytical ultracentrifuge illustrating the key components. (a) Absorbance detection system often used to monitor the concentration profiles of proteins and nucleic acids. (b) Refractive optical system used for precision measurement of concentration profiles. (c) Rotor that holds the sample cells. This is made of titanium and may be spun at speeds up to 60 000 rpm. (d) Cell containing the samples. As each cell passes an optical detector, (a) or (b), the concentration profile is obtained. (e) Electric motor that spins the rotor. (f) Detector camera for the refractive optical system. Drawing courtesy of Beckman Coulter, Inc.

Figure 3.

Concentration profile from a sedimentation equilibrium experiment conducted on the same protein as in Figure . For these refractometric data, the centrifuge was run at 30 000 rpm and 20°C. The protein is confined to the region between the meniscus, rm, and the base, rb, of the cell. The sample has been sedimenting for 18 h before acquiring this concentration profile. The system is at equilibrium, meaning the concentration profile is invariant with time.

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References

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Further Reading

Cole JL, Lary JW, Moody TP and Laue TM (2008) Analytical ultracentrifugation: sedimentation velocity and sedimentation equilibrium. In: Correia JJ, Detrich W and Han L (eds) Methods in Cell Biology, vol. 84, Chapter 6, pp. 143–179. Holland: Elsevier.

Laue TM and Stafford WF (1999) Modern applications of analytical ultracentrifugation. In: Stroud RM (ed.) Annual Reviews of Biophysics and Biomolecular Structure, pp. 75–99. San Francisco: Annual Reviews.

Lebowitz J, Lewis MS and Schuck P (2002) Modern analytical ultracentrifugation in protein science: a tutorial review. Protein Science 11: 2067–2079.

Schachman HK (1959) Ultracentrifugation in Biochemistry. New York: Academic Press. 272 pp.

Schuster TM and Laue TM (1994) Modern Analytical Ultracentrifugation: Acquisition and Interpretation of Data for Biological and Synthetic Polymer Systems. Boston: Birkhauser. 351 pp.

Van Holde KE (1985) Sedimentation. In: Physical Biochemistry, pp. 110–136. Englewood Cliffs, NJ: Prentice Hall.

Williams JW, Van Holde KE, Baldwin RL and Fujita H (1958) The theory of sedimentation analysis. Chemical Reviews 58: 715–806.

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Maxon Laue, Thomas(Apr 2010) Sedimentation. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002982.pub2]