Ultracentrifugation macromolecules

At high speeds greater than 40,000 rpm in the ultracentrifuge, macromolecules settle towards the rotor periphery. Under these conditions the sedimentation coefficient s, is determined from the speed of sedimentation divided by the angular acceleration. The sedimentation coefficient is related to molecular weight using Equation (4.15). 

Karibyants N, Dautzenberg H, Colfen H. Characterization of PSS/ PDADMAC-co-AA polyelectrolyte complexes and their stoichiometry using analytical ultracentrifugation. Macromolecules 1997 30 7803—7809. 

In order to understand polymer solution behaviour, the samples have to be characterised with respect to their molecular configuration, their molar mass and polydispersity, the polymer concentration and the shear rate. Classical techniques of polymer characterisation (light scattering, viscometry, ultracentrifugation, etc.) yield information on the solution structure and conformation of single macromolecules, as well as on the thermodynamic interactions with the solvent. In technical concentrations the behaviour of the dissolved polymer is more complicated because additional intramolecular and intermolecular interactions between polymer segments appear. 

An analytical ultracentrifugation method for determining the molecular mass, diffusion coefficient, and/or state of oligomerization of a macromolecule by conducting sedimentation conditions to establish an equilibrium distribution of the macromolecule from the meniscus to the bottom of the observation cell. 

Svedberg invented the ultracentrifuge and used it to demonstrate the existence of macromolecules. 

Schuck, P. (2000). Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys.J. 78(3), 1606-1619. 

Study of enzyme structure often means elucidation of protein structure. In the early stage of enzyme studies, protein was considered to be the heterogeneous macromolecule that could be never determined by methods available in those days. In 1926, Svedberg developed an ultracentrifugation technique to determine molecular weight of macro-... 

Many of the methods used to extract information related to the structure of macromolecules come from studying the behavior of isolated macromolecules in solution. These techniques are based primarily on the flow behavior in a velocity gradient, the rate of Brownian motion of a particle, or osmotic effects associated with the size of individual molecules. The techniques that have been employed to study size and shape of macromolecules most extensively include viscometry, light scattering, analytical ultracentrifugation, and electron microscopy. 

The size and shape of macromolecules in solution can be studied using two techniques termed equilibrium and velocity ultracentrifugation. These techniques use an ultracentrifuge to rotate solutions of macromolecules and place them under a centrifugal force to study their physical properties. The size and shape of the macromolecules can then be determined from the solution physical properties. The ultracentrifuge is equipped for direct measurement of the solution as it spins at high speed. 

Values of M and s determined by analytical ultracentrifugation are given in Table 4.4 for some biological macromolecules. From these values we can get an idea of the shape factor for a given biological macromolecule. 

Table 4.4. Determination of molecular weights and sedimentation coefficients for connective tissue macromolecules using ultracentrifugation reproduced from Silver, 1987...

Sedimentation in an ultracentrifuge is a powerful means for separating biological particles and large macromolecules. Sedimentation is the only static field (Sc) method other than electrophoresis (in all of its various forms) commonly used for multicomponent separations. 

Svedberg proves unambiguously the existence of macromolecules by means of the ultracentrifuge he also develops the first precise method for obtaining the molecular weight distribution... 

Steinberg IZ, Schachman HK (1966) Ultracentrifugation Studies with Absorption Optics, V, Analysis of Interacting Systems Involving Macromolecules and Small Molecules. Biochemistry 5(12) 3728... 

Pollet, R.J. (1985) Characterization of macromolecules by sedimentation equilibrium in the air-turbine ultracentrifuge, Methods Enzymol. 117, 3-27... 

Centrifugation can also be used to study macromolecule-ligand interactions, provided that ligand binding alters the sedimentation coefficient of the macromolecule. The analytical ultracentrifuge is well suited for quantitative studies of binding it is a... 

---