Ultracentrifugation sedimentation equilibrium

The apparent Mr of LCAT is approximately 65,000 by SDS-polyacryla-mide gel electrophoresis (A10, A16, C14, K15, M6, U7) or 59,000 by sedimentation equilibrium ultracentrifugation (C15). The difference in Mr obtained by these two methods may reflect the high carbohydrate content of LCAT (24% by weight), which would give an inaccurate estimate of Mr by SDS-polyacrylamide gel electrophoresis (C15). 

Horton, J. C., Harding, S. E., Mitchell, J. R., and Morton-Holmes, D. F. (1991). Thermodynamic non-ideality of dilute solutions of sodium alginate studied by sedimentation equilibrium ultracentrifugation. Food Hydrocoll. 5 125-127. 

Lovenberg, Buchanan, and Rabinowitz 65) showed that the molecular weight of C. pasteurianum ferredoxin is about 6000, based on sedimentation velocity and sedimentation equilibrium ultracentrifugation determinations and on amino acid analysis. The sedimentation coefficient, S2o,w was 1.4, and the partial specific volume, determined according to the method of Hvidt et al. 59) was 0.63, as compared to the value of 0.71 observed for most proteins. Similar investigations showed that ferredoxins from four other clostridia Lovenberg, Buchanan, and Rabinowitz 65)) and from a photosynthetic bacterium (Bachofen and Arnon 12)) also had a molecular weight of about 6000. 

Figure 3. Sedimentation equilibrium ultracentrifugation of peptidophospho-galactomannan (21). Solutions containing...

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. 

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). 

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. 

Basic Sedimentation Equilibrium Equation. Sedimentation equilibrium experiments are performed at constant temperature. The condition for sedimentation equilibrium is that the total molar potential, m, for all components i be constant everywhere in the solution column of the ultracentrifuge cell. Mathematically this can be expressed as... 

Evaluation of Mweq. Sedimentation Equilibrium Experiments and the Archibald Method. At constant temperature the condition for sedimentation equilibrium is that the total potential jZi for each associating species i (i = A, B, or AnBw) be constant at every radial position r in the solution column of the ultracentrifuge thus... 

The sedimentation equilibrium experiment requires much smaller volumes of solution, about 0.15 ml. With six-hole rotors and multichannel centerpieces (41) it is potentially possible to do fifteen experiments at the same time. For situations where the photoelectric scanner can be used one might (depending on the extinct coefficients) be able to go to much lower concentrations. Dust is no problem since the centrifugal field causes it to go to the cell bottom. For conventional sedimentation equilibrium experiments, the analysis of mixed associations under nonideal conditions may be virtually impossible. Also, sedimentation equilibrium experiments take time, although methods are available to reduce this somewhat (42, 43). For certain situations the combination of optical systems available to the ultracentrifuge may allow for the most precise analysis of a mixed association. The Archibald experiment may suffer some loss in precision since one must extrapolate the data to the cell extremes (rm and r6) to obtain MW(M, which must then be extrapolated to zero time. Nevertheless, all three methods indicate that it is quite possible to study mixed associations. We have indicated some approaches that could be used to overcome problems of nonideality, unequal refractive index increments, and unequal partial specific volumes. 

The molecular weight of the enzyme has remained remarkably constant from the bacteria to the higher mammals (Table VIII). The molecular weight from all species averages 36,500, and the deviations from this value fall within the experimental range of the methods used, in this case sedimentation equilibrium in the ultracentrifuge or electrophoresis of the SDS-enzyme-complexes on polyacrylamide gel. 

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