Ultracentrifugation, equilibrium

If a solution of a substance of low molecular weight is ultracentrifuged, equilibrium is established within a fairly short period of time, and there will be a density gradient in the solution. If a substance of high molecular weight is added to this solution it will float at the position at which its density is equal to the density of the solution. 

Figure 3.69. Concentration (wt %) dependence of the interaction parameter in the polystyrene- -cyclohexane system (iW == 163,000) at T =. 35 0, 45°C, and 6-5 C from light scattering (/) (Scholte, 1970b), the critical state (S) (Koningsveld et al., 1970b), and the ultracentrifuge equilibrium (.9) (Scholte, 1970b) [Reprinted from Th.G.Scholte. Europ. Polym. J. 6 (1970) 1063 1074. Copyright 1970 with kind permission of Eliievier Science — NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands]...

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. 

Winzor and coworkers have employed measurements of the Donnan distribution of small ions in dialysis equilibrium [14] to reinforce earlier evidence of charge-screening effects in polysaccharide anions [164,165]. These researchers used the absorption optical system of a Beckman XL-1 ultracentrifuge to monitor the distribution of ions in polysaccharide solutions... 

With this relationship for all samples was calculated from ninh This M is used for evaluating the reaction data. The ultracen rifuge (u.c measurements were carried out in a Spinco model E analytical ultracentrifuge, with 0.4% solutions in 90% formic acid containing 2.3 M KCl. By means of the sedimenta- ion diffusion equilibrium method of Scholte (13) we determine M, M and M. The buoyancy factor (1- vd = -0.086) necessary for tSe calculation of these molecular weights from ultracentrifugation data was measured by means of a PEER DMA/50 digital density meter. 

Physical separation methods can be based on equilibrium considerations, but the majority are not. Ordinary filtration is an example of a non-equilibrium, physical method and so is ordinary centrifugation— e.g.—the separation of a precipitate from the suspending liquid using an artificial gravity field. There are separation methods, which are called filtration which are not such as gel filtration. Ultracentrifugation in a salt gradient is a physical equilibrium method. 

Alternatively, in sedimentation equilibrium experiments, the ultracentrifuge is operated at slow speeds for longer time to permit the solute molecules to attain equilibrium between sedimentation and diffusion. If the sample is monodisperse, its concentrations cb c2 can be measured at 2 positions xh x2 in the cell. 

Binding of heme by isolated N-domain causes a change in sedimentation coefficient consistent with a more compact conformation and leads to the more avid association with the C-domain (125). Sedimentation equilibrium analysis showed that the Kd decreases from 55 pM to 0.8 pM (Fig. 5) (106). In addition, the calorimetric AH (-1-11 kcal/mol) and AS (-1-65 kcal/mol K) for the heme-N-domain-C-domain interaction and the AH (-3.6 kcal/mol) and AS (-1-8.1 kcal/mol K) derived from van t Hoff analysis of ultracentrifuge data for the interaction in the absence of heme indicate that hydrophobic interactions predominate in the presence of heme and a mix (e.g., hydrophobic and van der Waals forces) drives the interaction in the absence of heme. However, FTIR spectra (Fig. 6) indicate that little change in the secondary structure of domains or intact hemopexin occurs upon heme binding (104). 

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. 

SEC-MALLS and SEC-LALLS Coupled chromatography and light-scattering photometry that allows the determination of a number of important values along with chain length distribution, sedimentation equilibrium experiment Ultracentrifugation technique that allows chain length information to be determined. 

Analytical ultracentrifugation s Equilibrium sedimentation at different car-bohydrate protein ratios yields stoichiometry of complex... 

When mixtures of solvents of different density are used for polymer dissolution, an equilibrium is established during ultracentrifugation where the concentration of the solvent of higher specific gravity is increased at the bottom of the cell... 

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