Analytical ultracentrifugation: sedimentation velocity and sedimentation equilibrium

Abstract

Analytical ultracentrifugation (AUC) is a versatile and powerful method for the quantitative analysis of macromolecules in solution. AUC has broad applications for the study of biomacromolecules in a wide range of solvents and over a wide range of solute concentrations. Three optical systems are available for the analytical ultracentrifuge (absorbance, interference, and fluorescence) that permit precise and selective observation of sedimentation in real time. In particular, the fluorescence system provides a new way to extend the scope of AUC to probe the behavior of biological molecules in complex mixtures and at high solute concentrations. In sedimentation velocity (SV), the movement of solutes in high centrifugal fields is interpreted using hydrodynamic theory to define the size, shape, and interactions of macromolecules. Sedimentation equilibrium (SE) is a thermodynamic method where equilibrium concentration gradients at lower centrifugal fields are analyzed to define molecule mass, assembly stoichiometry, association constants, and solution nonideality. Using specialized sample cells and modern analysis software, researchers can use SV to determine the homogeneity of a sample and define whether it undergoes concentration-dependent association reactions. Subsequently, more thorough model-dependent analysis of velocity and equilibrium experiments can provide a detailed picture of the nature of the species present in solution and their interactions.

Figure 1

Basic analytical ultracentrifugation experiments. Simulations are for a protein of 50 kDa with a sedimentation coefficient of 4 S. A) SV experiment. Velocity sedimentation is usually performed using a two-sector cell and scans are recorded at fixed intervals during the run. The simulation is for a rotor speed of 50,000 RPM and scans are displayed at 20 minute intervals. B) SE experiment. Equilibrium measurements usually employ a six sector cell with three loading concentrations. The equilibrium concentration gradients are simulated for four rotor speeds ranging between 12,000–32,000 RPM, corresponding to values of ranging from ~ 0.8 to ~ 6 cm⁻². The 32,000 RPM scan is truncated at the base.

Figure 2

Typical workflow for a analytical ultracentrifugation analysis of an unknown sample. For details see the text.