Analytical ultracentrifugation (AUC) is a biophysical technique that characterizes macromolecules in solution by their sedimentation behavior under high centrifugal force. It determines molecular weight, hydrodynamic shape, binding stoichiometry, and equilibrium constants without requiring standards or column calibration.
Two complementary experiments are performed. Sedimentation velocity spins samples at high speed and monitors the radial movement of the boundary as molecules sediment. Sedimentation equilibrium uses lower speeds where sedimentation is balanced by diffusion, producing an exponential concentration gradient at equilibrium.
Sedimentation velocity AUC provides the sedimentation coefficient distribution. The sedimentation coefficient depends on molecular mass and frictional coefficient. The frictional coefficient reports on molecular shape. The diffusion coefficient is obtained from boundary spreading. The Svedberg equation relates these parameters. The analysis using continuous c(s) distribution models in programs such as SEDFIT accounts for diffusion-deconvoluted sedimentation coefficients.
Sedimentation equilibrium AUC provides molecular weight independent of shape. The equilibrium concentration gradient follows an exponential function of radial position. For a single ideal species, the molecular weight is determined from the slope of the ln c versus r plot. Multi-component analysis resolves mixtures and determines association constants for interacting systems.
Detection systems include absorbance optics, Rayleigh interference optics, and fluorescence detection. Absorbance detection is wavelength-specific and suitable for chromophoric samples. Interference detection measures concentration changes for all solutes and is more sensitive. Fluorescence detection achieves single-molecule sensitivity for labeled samples.
AUC applications include characterization of protein oligomerization, antibody aggregation, protein-nucleic acid interactions, lipoprotein heterogeneity, and nanoparticle size distribution, complementing methods such as dynamic light scattering. The technique is label-free and operates in native solution conditions. It is considered the gold standard for determining the hydrodynamic properties of macromolecules.
Sample requirements include optical transparency, appropriate buffer density and viscosity, and sufficient concentration for detection. Typical concentrations range from 0.1 to 10 mg/mL for absorbance detection.
