There are two main types of analytical ultracentrifuge experiments: sedimentation velocity and sedimentation equilibrium experiments. In sedimentation velocity experiments, the change in concentration distribution in the ultracentrifuge cell at high rotor speeds is recorded. A boundary is formed between solution and clear sol vent left behind and the rate of movement of the boundary per unit centrifugal fi eld yields the sedimentation coefficient (Equation 12.8), which will be a function of the size (molecular mass) and shape (based on friction properties) of the system. For heterogeneous systems the method will give a distribution of sedimentation coefficient.
In contrast, in sedimentation equilibrium experiments an equilibrium or steady state distribution of concentration is obtained. Here, lower rotor speeds are used and the centrifugal forces are countered by the back forces due to diffusion and no boundary is formed. Instead, after a period of time (usually at least several hours) an equilibrium is reached, and there will be no net movement of macromolecules, hence no friction forces: the pattern only depends on molecular mass. The two methods – sedimentation velocity and sedimentation equilibrium – provide complementary information about a macromolecular system. Sedimentation velocity has a high resolution, which is excellent for monitoring heterogeneity and aggregation and also for evaluating molecular conformation in solution. Sedimentation equilibrium is an ‘absolute’ method (not requiring standards) for molecular mass and (in heterogeneous systems) average molecular mass determination. For systems containing non-covalent assemblies, both methods can provide valuable information about the stoichiometry and strength of self-association reactions (i.e. the quaternary structure: protein dimerisation, tetramerisation, etc.) or interactions with other molecules.
Analytical ultracentrifugation is most often employed for:
• the determination of the purity (including the presence of aggregates) and oligomeric state of macromolecules, from recording the distribution of sedimentation coefficients from sedimentation velocity
• the determination of the average molecular mass, or distribution of molecular mass of solutes in their native state, from sedimentation equilibrium
• the examination of changes in the molecular mass of supramolecular complexes, using either sedimentation velocity or sedimentation equilibrium (or both)
• the detection of conformation and conformational changes using sedimentation velocity
• ligand-binding studies.
Since the mass of one molecule is extremely small, when researchers refer to the ‘molecular mass’ of a molecule, they really mean the molar mass M which describes the mass of 1 mol (= 6.023 × 1023 molecules) of macromolecules, in units of g mol –1 , or, equivalently, the relative molecular mass M r , which is the mass of a macromolecule per one twelfth of the mass of a carbon-12 atom. Molar mass and relative molecular mass are numerically the same, but being a relative measure, Mr has no units.
For a list of references outlining the applicability of ultracentrifugation to the characterisation of macromolecular behaviour in solution. In addition, manufacturers of analytical ultracentrifuges offer a large range of excellent brochures on the theoretical background of this method and its specific applications available. These introductory texts are usually written by research biochemists and are well worth reading to become familiar with this field.
