Sedimentation Equilibrium in a Density Gradient

Isopycnic zone ultracentrifugation, on the other hand, deals with the preparative analog to equilibrium sedimentation in a density gradient. Here, large density gradients are used and one waits until equilibrium is attained. Both band and isopycnic zone ultracentrifugation can be carried out with very small solute concentrations. These methods have become, therefore, particularly important in the separation of biological macromolecules (biopolymers). Both methods are also described in the literature under a variety of other names. 

There are two principal methods that are suitable for quantitative determination of the constitutional heterogeneity fractionation and equilibrium sedimentation (equilibrium centrifugation) in a density gradient. In fractionation, the different solubilities of the polymers of various com-... 

Figure 9-14. Diagrammatic representation of the establishment of a sedimentation equilibrium in a density gradient c is the concentration of the macromolecular solute).

J. Vinograd and J. E. Hearst, Equilibrium sedimentation of macromolecules and viruses in a density gradient, Fortschr. Chem. Org. Naturstoffe 20, 372 (1962). 

To study the biochemical properties of membrane proteins, we use equilibrium sedimentation in a sucrose density step gradient (0.5 M, 2.0 M, 2.5 m) to purify the membrane fraction (Figure 32.1) following a procedure first developed by Strand et al. (1994). Protocol 32.1 describes this membrane-extraction procedure. Protocol 32.2 includes two useful methods to determine the membrane association of a protein of interest (1) alkaline wash and (2) Triton X-114 phase separation. The integrity of the Drosophila mem-... 

Equilibrium sedimentation technique working with a multi-component solvent forming a density gradient in a centrifugal field... 

Particles in the gradient may be separated on the basis of sedimentation rate a sample introduced at the top of the preformed gradient setdes according to density and size of particles, but the run is terminated before the heaviest particles reach the bottom of the tube. If the density of all the particles lies within the range of the density limits of the gradient, and the run is not terminated until all particles have reached an equilibrium position in the density field, equilibrium separation takes place. The steepness of the gradient can be varied to match the breadth of particle densities in the sample. 

A technique which has not yet been applied to polysaccharides is ultra-centrifugation with a density gradient in the cell. In this method, the molecules sediment until they come to equilibrium with solvent of their own density, and concentration bands are set up corresponding to the different polymeric species. This technique may provide a sensitive test for homogeneity. 

Figure 9.I. A. Profile of Ciona DNA (molecular size > 100 kb), as obtained by analytical ultracentrifugation to sedimentation equilibrium in a CsCl gradient. The 1.742 g/cm peak is the peak of the marker DNA from phage 2C. B. Fit of analytical profile of Ciona DNA (curve) with a gaussian curve (dotted line). This analysis was performed using the MacCurveFit program. The GC scale was obtained from the buoyant density values using the relationship of Schildkraut et al. (1962). (From De Luca et al., 2002).

Up to now, it has been assumed that the solvent in the sedimentation experiment consists of a single component. If, however, the solvent system consists of a mixture of two substances of widely different densities (e.g., CsCl in water or mixtures of benzene and CBr4), the solvent components will sediment to different extents. At equilibrium, the solvent system possesses a density gradient. One density p , applies at the bottom of the cell, and another, pm, at the meniscus. The density of the solute, pi, should lie between these two densities (pm < pi < pt). The macromolecules will then sediment from the meniscus toward the base of the cell and float from the base toward the meniscus (Figure 9-14). At equilibrium, the macromolecules will take up a position (designated by ) at which the density pg exactly corresponds to the density of the macromolecule in solution (pg = pi 1 / V2). This position is at a distance r from the center of rotation. 

SEdIMENtatIOn-FLOtation focusing fff In SFFFFF solute particles or macromolecules sedimenf or float in the density gradient of a liquid phase according to the difference between the local density of the liquid and that of the particle or macromolecule. At quasi-equilibrium, the solute species are focused in a thin layer at the position where the density of the environment is the same as the solute density. 

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