Analytical ultracentrifugation equilibrium

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. 

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. 

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. 

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

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. 

Using Analytical Ultracentrifugation (AUC) to Measure Global Conformational Changes Accompanying Equilibrium Tertiary Folding of RNA Molecules... 

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 energetics of protein association can be studied by a variety of experimental techniques,17 each of which permits measurements of equilibrium or kinetic values in a certain range. Widely used techniques include isothermal titration calorimetry, surface plasmon resonance measurement, stopped flow kinetics, optical spectroscopy, MS, and analytical ultracentrifugation. The techniques differ in their requirements (e.g., amount of protein, labeling with fluorophores, attachment to sensor surfaces, and the environment provided by the experimental set up) and therefore in their applicability to individual cases. Different techniques can also give quite different values for what might be expected to be the same quantity. For example, association rates measured by surface plasmon resonance, with one protein immobilized on a surface, are usually different from those measured for the two proteins in solution and under otherwise similar conditions. 

The purified hemagglutinin was homogeneous by gel filtration and immuno-electrophoresis, and in the analytical ultracentrifuge.63,560,561 A molecular weight of 79,000 was determined by the sedimentation equilibrium method561,569 (100,000 by sedimentation and velocity measurements,83 and 53,000 by gel filtration563). 

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). 

Here we describe studies of the interaction of interleukin-6 (IL-6) with a soluble form of its cell surface receptor (sIL-6R). A procedure utilising a competition approach is presented which allows the determination of the equilibrium constant in solution thus avoiding any potential problems associated with deviation in kinetic characteristics upon surface immobilisation. In addition, binding characteristics of stable monomeric and dimeric forms of IL-6 are presented to demonstrate both the drastic influence of solute multivalency on kinetic and equilibrium properties and the importance of auxiliary techniques such as analytical ultracentrifugation for the interpretation of SPR data. 

Sedimentation equilibrium analytical ultracentrifugation of isolated ZDD was used to determine its solution molecular weight. These experiments revealed the presence of a single species in solution corresponding to the molecular mass of the dimeric ZDD fragment. Global analysis of the equilibrium data using equation 1... 

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