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INTRODUCTION TO

ANALYTICAL

ULTRACENTRIFUGATION

ii

INTRODUCTION TO ANALYTICAL ULTRACENTRIFUGATION

GREG RALSTON

DEPARTMENT OF BIOCHEMISTRY

THE UNIVERSITY OF SYDNEY

SYDNEY, AUSTRALIA

iii iv

CONTENTS

About the Author

About this Handbook

Glossary

Recommended Reading

Analytical Ultracentrifugation and Molecular Characterization The Unique Features of Analytical Ultracentrifugation

Examination of Sample Purity

Molecular Weight Determination

Analysis of Associating Systems

Conformation Changes

Ligand Binding

Sedimentation of Particles in a Gravitational Field

Instrumentation

Rotors

Cells

Boundary forming cells

Band forming cells

Methods of Detection and Data Collection

Refractometric Methods

Schlieren

Rayleigh interference optics

Absorbance

Sample Preparation

Multiple Boundaries

Determination of s

Solvent Effects

Concentration Dependence

Radial Dilution

Analysis of Boundaries

Self-Sharpening of Boundaries

Tests for Homogeneity vi

vii viii x 1 3 3 3 5 6 7 8 11 11 11 14 14 15 15 15 17 18 20 22
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Speed Dependence

Primary Charge Effect

Association Behavior

Band Sedimentation

Active Enzyme Sedimentation

Diffusion

Sedimentation Equilibrium

Subunit Structure

Heterogeneity

Nonideality

Association Reactions

Determination of Thermodynamic Parameters

Detergent-Solubilized Proteins

Behavior in "Crowded" Solutions

Archibald Approach-to-Equilibrium Method

Density Gradient Sedimentation Equilibrium (Isopycnic

Sedimentation Equilibrium)

The Future

References

Index

FIGURES

Figure 2

Double-sector centerpiece

Figure 3

Comparison of the data obtained from the schlieren, interference, photographic absorbance, and photoelectric absorbance optical systems

Figure 4

Schematic diagram of the optical system of the Beckman

Figure 5

Movement of the boundary in a sedimentation

velocity experiment with a recombinant malaria antigen protein

Figure 6

Plot of the logarithm of the radial position, r

bnd , of a sedimenting boundary as a function of time for recombinant dihydroorotase domain protein 33
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Figure 7

Concentration dependence of the sedimentation

Figure 8

thymus DNA fragments

Figure 9

The primary charge effect

Figure 10 Concentration-dependent increase in weight average Figure 11 Schematic appearance of a bimodal boundary for a hypothetical monomer-tetramer association reaction Figure 12 Spreading of the boundary with time in a diffusion experiment with dextran

Figure 13

Figure 14 Schematic representation of sedimentation equilibrium Figure 15 Schematic representation of the meniscus in a centrifuge cell Figure 16 Sedimentation equilibrium distribution of two different solutes Figure 17 Decrease in apparent molecular weight with Figure 18 Sedimentation equilibrium analysis of the selfassociation of a DNA-binding protein from B. subtilis Figure 19 Diagnostic plots for assessing the self-association of

Ά-lactoglobulin C

Figure 20 Sedimentation equilibrium analysis of human spectrin TABLE

Table 1

for Common Biological Macromolecules 27
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vii

ABOUT THE AUTHOR

Greg Ralston is an Associate Professor in the Department of Biochemistry at the University of Sydney. His research interests center on understanding the interactions within and between proteins. He has a degree in Food Technology from the University of NSW, and a Ph.D. from the Australian National University, where he studied with Dr. H. Carlsberg Laboratory in Denmark, he studied with Prof. J. W. Williams at the University of Wisconsin, where he began his research on the self-association of the protein spectrin from erythrocyte membranes. This research has continued at the University of Sydney, where he has built up a modern analytical ultracentrifuge facility. viii

ABOUT THIS HANDBOOK

ultracentrifugation, is intended for scientists who are contemplating the use of this powerful group of techniques. The goals of this little book are: to introduce you to the sorts of problems that can be solved through the application of analytical ultracentrifugation; to describe the different types of experiments that can be performed in an analytical ultracentrifuge; to describe simply the principles behind the various types of experiments that can be performed; and to guide you in selecting a method and conditions for a particular type of problem. ix

GLOSSARY

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