Sedimentation Velocity Analysis

In this technique, the particle moves toward an equilibrium position, but the experiment is terminated before equilibrium occurs. The change in velocity of the particle is interpreted using kinetic equations. Consider a simple two-component system, 

A plot of In r against t (Eq. 13.11) or two measurements of boundary position (Eq. 13.12) can be used to determine the value of s. Usually, the sedimentation coefficient is expressed in Svedberg units, where 1 S = 10-13 s. 

The value can be used to calculate the molecular weight of a macromolecule, using Eq. 13.13  

The diffusion coefficient indicates the extent of boundary broadening during the experiment, but is not easy to determine, limiting the use of this method. 

In order to use Eq. 13.15 to obtain molecular weights, experimentally obtained, v and D values must be corrected to ideal conditions. The observed, v and D values are dependent on solution conditions as well as temperature. It is possible to convert an observed 5 value to a standard value for a solution with the density and viscosity of water at 20 °C using the following equation 13 

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Tung,L.H., Runyon,J.R. Molecular weight distribution of standard polystyrene samples by GPC and by sedimentation velocity analysis. J. Appl. Polymer Sci. 17, 1589-1596 (1973). 

The data for the narrow distribution polystyrene C-105 are given to provide a standard of comparison for the numbers reported for the experimental polymers. The GPC results indicate a broader distribution than that obtained by sedimentation velocity analysis. Such differences between GPC results and the more traditional measurements have been reported previously (I). 

Figure 1 Sedimentation velocity analysis of the interaction between bisANS and bacteriophage P22 coat protein. Sedimentation was carried out at 56,0(X) rpm at 20 °C in a Beckman Model E centrifuge equipped with a video-based on-line Rayleigh optical system. (TOP) Coat protein alone (cq = 0.4 mg/ml t = 5348 sec) (BOTTOM) Coat protein in the presence of 60 (iM bisANS (A) Co = 0.5 mg/ml t = 5339 sec (B) 0.2 mg/ml t = 5343 sec). The error bars are the standard error of the mean propagated from the averaging process.

Figure 4. Sedimentation Velocity Analysis of ZDD. A, Primary data collected at 1 mg/ml (10 scans). B, Apparent sedimentation coefficient distribution function, g(s ) versus s. The error bars represent the standard error of the mean. The solid line is the fit to equation 4. Apparent s, D, and Ms,D values were calculated as described.

When rat pancreatic polynucleosomes were poly(ADP-ribosylated) with purified calf thymus poly(ADPR) polymerase and examined by electron microscopy a relaxation of their native zigzag structure was observed, even at high ionic strengths they showed a close resemblance to chromatin depleted of histones HI. The relaxed state of poly(ADP-ribosylated) polynucleosomes was also confirmed by sedimentation velocity analysis [19, 20]. Locally relaxed regions can also be generated within poly-nucleosome chains by the activity of their intrinsic poly(ADPR) polymerase and appeared to be correlated with the formation of hyper(ADP-ribosylated) forms of histone HI and an increase of DNA polymerase activity [21]. The posttranslational transitory modifications of histones are potential modulators of chromatin stmcture. This may be involved in DNA transcription, replication, and repair. 

In order to investigate the modification of DNA accessibility in chromatin structures relaxed by ADP-ribosylation, the DNA polymerase a activity has been determined [22]. As shown in Table 1, this activity is more than twofold higher in polynucleosomes ADP-ribosylated for 25 min than in control polynucleosomes. Moreover, this increase in template capacity is correlated with the ADP-ribosylation induced relaxation time course as demonstrated by electron microscopy and sedimentation velocity analysis. 

In analytical ultracentrifuges, molecular properties can be modeled through sedimentation velocity analysis or sedimentation equilibrium analysis. In sedimentation velocity analysis, concentrations and solute properties are modeled continuously overtime. Sedimentation velocity analysis can be used to determine the macromolecule s shape, mass, composition, and conformational properties. During sedimentation equilibrium analysis, centrifugation has stopped and particle movement is based on diffusion. This allows for modeling of the mass of the particle as well as the chemical equilibrium properties of interacting solutes. 

By carrying out the sedimentation velocity analysis not only in H2O but also in D2O and in a 1 1 H20 D20 mixture (H2O/D2O analysis), both the PSD and information on the density (and thus chemical uniformity) of the individual particle fractions may be obtained. 

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