Molecular-weight centrifugation equilibrium

Problem 8.8 Molecular weight from equilibrium measurements in centrifugal or gravity fields... 

An alternative method sometimes used to determine molecular weights of macromolecules is sedimentation equilibrium. In the previous example, using the Svedberg equation, the sample is rotated at a rate sufficient to sediment the particles. Here, the sample is rotated at a lower rate, and the particles sediment until they reach an equilibrium position at the point where the centrifugal force is equal to the frictional component opposing their movement (see Equation 7.6). The molecular weight is then calculated using Equation 7.12. 

Centrifugation is used for two basic purposes (1) as a preparative technique to separate one type of material from others and (2) as an analytical technique to measure physical properties (e.g., molecular weight, density, shape, and equilibrium binding constants) of macromolecules. The sedimentation constant, s, of a protein is a measure of its sedimentation rate. The sedimentation constant is commonly expressed in svedbergs (S) 1 S = 10 seconds. 

In the sedimentation equilibrium technique the centrifuge is operated at a lower speed—e.g., about 8000 rpm for a molecular weight of 60 000. When the centrifuge... 

An alternative method of using the ultracentrifuge to measure molecular weights is to allow the distribution of particles to reach equilibrium. As sedimentation occurs in the ultracentrifuge a concentration gradient is established, and this will cause the molecules to diffuse in the opposite direction. Eventually the system reaches a state of equilibrium at which the rate with which the solute is driven outwards by the centrifugal force just equals the rate with which it diffuses inwards under the influence of the concentration gradient. 

In equilibrium centrifugation of an ideal polymer, the molecular weight is related to the ratio of the concentrations C2/C1, at two points in the centrifuge cell at distances. Ti and from the axis of rotation by the equation... 

In sedimentation equilibrium experiments, the centrifuge is run at slower speeds, and the opposing forces of sedimentation and diffusion are allowed to come to equilibrium. The resulting concentration distribution over the length of the cell is used to determine the molecular weight of the protein, the oligomeric state of the protein (e.g., monomers versus dimers or tetramers, etc.), and the association constant of self-association (e.g., dimerization, tetramerization, etc.). 

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