Analytical Centrifugation: Equilibrium Approach
互联网
886
- Abstract
- Table of Contents
- Figures
- Literature Cited
Abstract
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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryTable of Contents
- Theory
- Strategy
- Data Analysis and Interpretation
- Literature Cited
- Figures
- Tables
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryMaterials
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online LibraryFigures
-
Figure 20.3.1 A schematic representation of an analytical ultracentrifuge. The rotor (C ) has holes through it to hold sample containers commonly called cells (D ). Each cell (see inset) consists of a centerpiece (G ) with open‐sided chambers called channels (H ) to hold the liquid samples. There are two channels per sample, one containing the protein in its solvent and an adjacent one (not shown) containing only solvent to serve as an optical reference. The centerpiece, in turn, is sealed between windows (F ) to permit the passage of light through the channels, thus allowing the cell contents to be viewed. Centerpieces are made out of a variety of tough inert materials. Depending on the type of experiment that will be performed, centerpieces can hold one, three, or four samples each. Rotors can hold either four or eight cells. As the rotor spins, each cell passes through the optical paths of two different detectors. The absorbance detector uses a pulsed xenon lamp (A ) to provide a burst of light when a cell is aligned in the beam. The absorbance detector (E ) uses a narrow slit and photomultiplier tube to determine the light intensity after the beam has passed through the sample. The slit is moved radially by a motor so that the absorbance profile, called an absorbance scan, can be determined. The second detector determines the refractive index difference between the sample and reference channels at each radial position. The light source (B ) uses a laser diode to produce two radially directed narrow stripes of light, one that passes through the sample channel and one that passes through the reference channel. These two stripes of light are brought together to produce an interference image (Fig. ) in which the difference in the refractive index at each radial position is displayed as the vertical displacement of a set of fringes. View Image -
Figure 20.3.2 A Rayleigh interference refractive optical system image of one channel of a six‐channel centerpiece (Fig. ). This optical system provides an image in which the concentration at each radial position is represented by the vertical displacement of a set of equally spaced horizontal fringes. The center of rotation is to the left and the edge of the rotor is to the right. An air/liquid meniscus forms at the top of the channel, one in the reference sector and one in the sample sector. As slightly different volumes of liquid are used to fill these two sectors, the menisci do not appear at exactly the same radius. If the two menisci do become superimposed over the course of an experiment, there is a leak between the sample and reference sectors, indicating that the centerpiece is scratched and needs polishing. The sample is a protein at sedimentation equilibrium, where the flux of molecules towards the cell bottom due to sedimentation is exactly balanced by the flux of molecules towards the meniscus due to diffusion. The balance of fluxes yields an exponential concentration distribution. The base of the cell is formed by FC‐43, a clear fluorocarbon liquid with a refractive index very close to that of water. View Image -
Figure 20.3.3 Flow chart of the protocol used to prepare a protein solution for analysis. View Image -
Figure 20.3.4 Descriptions of centerpieces commonly used for sedimentation equilibrium analysis under operating conditions usually employed. View Image
Videos
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 | |
| ftp://rasmb.bbri.org | |
| Site for MATCH program (David Yphantis and Jeff Lary), used for monitoring minimized average differences in concentration profiles over time. | |
| Tom.Laue@unh.edu | |
| Contact for Center to Advance Molecular Interaction Science (CAMIS), for assistance with data analysis. | |
| RASMB@rasmb.bbri.org | |
| Contact for the ultracentrifuge users group at Boston Biomedical Research Institute, for assistance with data analysis. | |
| http://www.bbri.org/RASMB/rasmb.html | |
| 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. |
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library
