Ultracentrifugation experiments, analytical

Rasa M, Meier MAR, Schubert US (2007) Transport of guest molecules by unimolecular micelles evidenced in analytical ultracentrifugation experiments. Macromol Rapid Commun... 

In principle, there are two forms of analytical ultracentrifuge experiment sedimentation equilibrium and sedimentation velocity. These can provide different, and to some extent complementary, information about the samples under study (Byron 1996). 

Other optical techniques have also been used to monitor analytical ultracentrifugation experiments. These included refractive index (Schlieren optics) as well as interference patterns produced using Rayleigh- or Lebedev-type optics.11 In both cases, data are acquired using photographic or video recording equipment. Examples of data obtained with these different optical systems are shown in Figure 13.11. 

Analytical ultracentrifugation experiments determine shape parameters and the average molecular weight of large molecules in solution, and can thus be used to deduce the stoichiometry of the complexes between hevein domains and... 

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. 

In addition to the determination of molar mass distributions and various molar mass averages there are many experiments, requiring sometimes sophisticated data evaluation, that can be carried out with an analytical ultracentrifuge. Examples are the analysis of association, the analysis of heterogeneity, the observation of chemical reactions, and protein characterization, to mention only a few. A detailed discussion is beyond the scope of this article, but there is excellent literature available [77-79,81,87-89]... 

The schlieren system of optics is an analytical method that is particularly well suited to following the location of a chemical boundary with time. It is routinely employed in ultracentrifuges and also in electrophoresis experiments, as we see in Chapter 12. Schlieren optics produces an effect that depends on the way the refractive index varies with position, that is, the refractive index gradient rather than on the refractive index itself. Therefore, the schlieren effect is the same at all locations along the axis of sedimentation, except at any place where the refractive index is changing. In such a region, it will produce an optical effect that is proportional to the refractive index gradient. The boundary between two layers is thus per-... 

The instrument routinely used in our lab for AUC experiments is the Beckman XL-I analytical ultracentrifuge. We exploit the UV absorption property of RNA molecules at 260 nm to employ the absorbance optics built in the centrifuge. The optical system of a Beckman analytical ultracentrifuge is shown schematically in Fig. 10.1A. The instrument is... 

In this section, we indicate the general factors that an experimentalist should consider while setting up a cation-mediated equilibrium RNA folding experiment, provide a stepwise protocol for sample preparation and outline the data collection procedure using the absorption optics of the analytical ultracentrifuge. 

The most important distinction between the two structures is that only the helical model is compatible with all of the published structural data, including results obtained from limited proteolysis [81,83], chemical cross-linking [83], analytical ultracentrifugation [81], and mutant complementation [84,85]. Although the double helix provides a useful model with good predictive power, it will no doubt be subjected to further investigation by, for example, atomic force microscopy or X-ray crystallography. Such experiments should refine the structural information on PKSs, and point the way toward the productive modification and immobilization of the synthases. 

The tertiary structure of retrocyclin-2 in a membrane-mimicking environment was determined recently and consists of a well-defined (3-hairpin braced by the three laddered disulphide bonds.193 Analytical ultracentrifugation and NMR diffusion experiments indicated that retrocyclin-2 self-associates to form a trimer. Self-association is an important feature of some antimicrobial peptides but the role of this self-association in the mechanism of action of the retrocyclins is yet to be determined. 

S-FFF has been compared with analytical ultracentrifugation (AUC) with respect to the fractionation of a 10-component latex standard mixture with narrow particle size distribution, known diameters (67-1220 nm) and concentration [ 127]. With an analytical ultracentrifuge, the particle sizes as well as their quantities could be accurately determined in a single experiment whereas in S-FFF deviations from the ideal retention behavior were found for particles >500 nm resulting in smaller particle size determination in the normal as well as in the programmed operation. It was concluded that, without a modified retention equation which accounts for hydrodynamic lift forces and steric exclusion effects, S-FFF cannot successfully be used for the size characterization of samples in that size range. 

With respect to the virtually very low sample loads, one could argue that the determined diffusion coefficient is at infinite dilution. However, there is no possibility in FFF techniques to perform a safe extrapolation to infinite dilution as in analytical ultracentrifugation or dynamic light scattering. In addition there is a severe problem for all particles deviating from the spherical shape (see Sect. 4.2.3 for a detailed discussion), as the evaluation of D from the FFF experiments using the conventional theory is inappropriate. 

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