Analytical Ultracentrifugation

Analytical Ultracentrifugation – Applications Determine sample purity Characterize assembly and disassembly mechanisms of biomolecular complexes Determine subunit stoichiometries Detect and characterize macromolecular conformational changes Measure equilibrium constants and thermodynamic parameters for self- and hetero-associating systems  characterize the solution-state behavior of macromolecules under various conditions

Analytical Ultracentrifugation – Design analytical ultracentrifuge = preparative ultracentrifuge + optical detection system  measure sample concentration inside the centrifuge cell during or after sedimentation centrifugation parameters and data acquisition under computer control  experiments lasting many days performed with minimal operator intervention

Analytical Ultracentrifugation – Design http://www-bioc.rice.edu/bios576/AU/AU%20Page_files/image022.jpg

Analytical Ultracentrifugation – Design: Optical systems Absorbance optical system:  measurement of sample concentration at wavelengths from 200 to 800 nm  detection of macromolecules containing strong chromophores Rayleigh interference optical system:  measurement of sample concentration based on refractive index changes  analyze macromolecules lacking intense chromophores (eg, polysaccharides) and samples that contain strongly absorbing buffer components (eg, ATP/GTP, DTToxidized)

Cindy Horwedel 21.01.2008

1-Analytical Ultracentrifugation – Sedimentation velocity experiments 21.01.2008

1-Analytical Ultracentrifugation – Sedimentation velocity experiments Meniscus of solvent Sedimentation front Meniscus of solution Plateau conc. Cell radius Concentration of solute Modified from http://www.kolloidanalytik.de/uz/sed/uzsedhr.gif

Experimentally, the data is gathered at high rotor speeds where the sedimentation transport dominates the diffusion. One measures a molecule's rate of transport from the top to the bottom of the cell. A sedimentation velocity experiment requires approximately 500 µg of sample protein in a volume of 1 ml.

1-Analytical Ultracentrifugation – Sedimentation velocity experiments Hydrodynamic information Experimentally determined parameters: Sedimentation coefficient s Diffusion constant D or friction factor f Molecular mass M Estimation of the molecule’s shape in solution

2-Sedimentation Equilibrium provides thermodynamic information Experimentally, the data are collected at slower rotor speeds so that the sedimentation and diffusion forces can balance. At sedimentation equilibrium, these forces are equal in magnitude, but opposite in direction, and the molecule is exponentially distributed across the cell. There is no longer any net transport of molecules in the system. All shape factors are thus cancelled out, yielding the molecular weight. A typical sedimentation equilibrium experiment requires 300 µl of sample at an OD of 0.8 at the wavelength of interest (usually 280 nm). This is generally on the order of 200-400 µg of total protein, but varies greatly depending on your protein's molar extinction coefficient.

21.01.2008

21.01.2008

Analytical Ultracentrifugation – Examples of Applications Sedimentation velocity Biomolecular Shape Biomolecular Conformational Changes Assembly and Disassembly of Biomolecular Complexes Molecular Mass and Subunit Stoichiometry Equilibrium Constants for Self-Associating Systems Sedimentation equilibrium Equilibrium Constants for Molecules