Ultracentrifugation sedimentation velocity

If the macromolecules are forced by some external agency to flow with a velocity V, light scattering can be used to measure this velocity. There are several possible examples of this (a) macromolecules suspended in a fluid which is in uniform motion with velocity Y, (b) macromolecules falling at their terminal velocities in a viscous solvent under the action of gravity (sedimentation velocity), (c) macroions accelerated to a terminal velocity by an externally imposed electric field (electrophoresis), and (d) macromolecules accelerated to their terminal velocity in an ultracentrifuge (sedimentation velocity). 

A protein was dissolved in buffer at pH 3.2 and was studied in the ultracentrifuge sedimentation velocity experiment. After the boundary was developed, five pictures were taken at intervals of 16 min. A typical picture with labels in a photographic plate is shown in the following sketch ... 

Apart from tliese mainstream metliods enabling one to gain a comprehensive and detailed stmctural picture of proteins, which may or may not be in tlieir native state, tliere is a wide variety of otlier metliods capable of yielding detailed infonnation on one particular stmctural aspect, or comprehensive but lower resolution infonnation while keeping tlie protein in its native environment. One of tlie earliest of such metliods, which has recently undergone a notable renaissance, is analytical ultracentrifugation [24], which can yield infonnation on molecular mass and hence subunit composition and their association/dissociation equilibria (via sedimentation equilibrium experiments), and on molecular shape (via sedimentation velocity experiments), albeit only at solution concentrations of at least a few tentlis of a gram per litre. 

The ultracentrifuge has been used extensively, especially for the study of biopolymers, and can be used in several different experimental modes to yield information about polymeric solutes. Of the possible procedures, we shall consider only sedimentation velocity and sedimentation equilibrium. We shall discuss these in turn, beginning with an examination of the forces which operate on a particle setting under stationary-state conditions. 

With sedimentation velocity we measure the change in solute distribution across a solution in an ultracentrifuge cell as a function of time. An example of such a change is given in Fig. 2a for potato amylose [29]. 

A nice example for the utilization of scaling laws in sedimentation velocity runs with the analytical ultracentrifuge has been published by Machtle and Borger [78]. For the polyelectrolyte sodium polystyrene sulfonate (NaPSS) in 0.5 molar NaCl solution they found a scaling law for the sedimentation coefficient at... 

Equations suitable for simulation of molecular weight distributions for any initial distribution and chosen values of G(scission) and G(crosslinking) have been developed and demonstrated. The molecular weight distributions may be obtained by GPC (with the limitation of changes in relative hydrodynamic volumes) and by sedimentation velocity in the ultracentrifuge. 

These solutions have been examined in sedimentation velocity runs on the analytical ultracentrifuge (31). Beyond 0.5 base equivalent per mole of iron a fairly narrow sedimentation peak developed. The sedimentation coefficient, 7 1 S, was essentially constant up to 2.5 base equivalents per mole of iron, although the area under the peak increased with increasing degree of hydrolysis. Apparently, then, hydrolysis of ferric nitrate beyond the reversible equilibrium region produces increasing amounts of a fairly discrete high polymer whose size is constant. 

Sedimentation velocity measurements were made on a Beckman-Spinco Model E ultracentrifuge equipped with RTIC. The sedimentation coefficients were not measured as a function of concentration for... 

The ultracentrifuge can be used to separate fractions of different molecular weights in a mixture of solutes and to determine the molecular weight of a solute. Two different approaches can be taken. In the sedimentation velocity approach, the change in the concentration profile with time is determined during the centrifugation process. This nonequilibrium process requires a knowledge of diffusion rates and is not based directly on thermodynamics. We will leave a discussion of this process to other texts. 

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

The dissociation of AMDH was also followed by sedimentation velocity measurements (Pundak et al, 1981) and neutron scattering (Zaccai et al, 1986b). Samples of AMDH were incubated at various NaCl concentrations at 20°C and aliquots were withdrawn at various times and analyzed by ultracentrifugation. Dissociation of the enzyme was visualized by the appearance of a slower sedimenting boundary (Fig. 1). The relative amounts of the protein in both boundaries could be measured and the dissociation rates could be calculated. These... 

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