Sedimentation Equilibrium Analysis

In this type of experiment, particles are allowed to move until they arrive at their equilibrium positions. This means that sedimentation proceeds until sedimentation forces are balanced by diffusion. There are several approaches to this technique. One of these is to scan the cell, to obtain the absorbance profile at different times this allows calculation of concentration as a function of distance from the rotation axis (dc/dr). The following expression is then used to calculate the particle mass  

It is possible to use this technique to study more complex systems, including poly-disperse samples in which interactions occur between several different components. Details regarding the mathematical methods can be found in Schachman.15 

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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). 

Fig. 5. Sedimentation equilibrium analysis of hemopexin domain interactions. Mixtures of N- and C-domain were centrifuged to equilibrium at 25 C in the absence (upper panels) and presence (lower panels) of heme. Nonlinear fitting procedures were performed to obtain apparent values, and residuals of the fits are shown in the top portion of each panel (106).

The determination that the low MW, acetylated aspen lignins examined actually fit universal calibration, however, must be deferred to future studies comparing these data to results from LALLS and sedimentation equilibrium analysis (if possible). 

The extent of the dissociation in 8 M urea can, moreover, not be estimated from the electrophoretically observed hybrid formation between wheat germ (isoenzyme II) SOD and respectively yeast and bovine SOD. BESOD did namely not dissociate in 8 M urea at 25 °C for at least 72 h, as indicated by sedimentation equilibrium analysis (Af. 32,000). The reactivity of Cys-6 and of the histidine residues was not increased either in that medium... 

Some of the physicochemical parameters of the enzyme, as determined on the Kunitz preparation (S), are summarized in Table I. Sedimentation equilibrium analysis of molecular weight carried out on the enzyme crystallized from ammonium sulfate gave a value of 71,000 (5), in fair agreement with the value of 63,000 obtained by sedimentation velocity measurements on the Kunitz preparation (10). The enzyme has been reported to dissociate into subunits in the presence of sodium dodecyl... 

Figure 13 illustrates the use of sedimentation equilibrium analysis to assess, in a complementary manner to SEC (see Section 13.2.5.3, Figures 11 and 12), whether the oligomerization state of a model coiled coil was responsive to single amino acid substitutions at one a position along the amphipathic a-helical strands. 

Figure 13 Sedimentation Equilibrium Analysis of Two-Stranded a-Helical Coiled-Coil Analogues N19a(ox) and L19a(ox)l491a bc...

The mass of a protein can be directly determined by sedimentation equilibrium, in which a sample is centrifuged at relatively low speed so that sedimentation is counterbalanced by diffusion. The sedimentation-equilibrium technique for determining mass is very accurate and can be applied under nondenaturing conditions in which the native quaternary structure of multimeric proteins is preserved. In contrast, SDS-polyacrylamide gel electrophoresis (Section 4.1.4) provides an estimate of the mass of dissociated polypeptide chains under denaturing conditions. Note that, if we know the mass of the dissociated components of a multimeric protein as determined by SDS-polyacrylamide analysis and the mass of the intact multimeric protein as determined by sedimentation equilibrium analysis, we can determine how many copies of each polypeptide chain is present in the multimeric protein. 

The liver acid phosphatase III thus isolated had a molecular weight of 14,000 daltons as determined by filtration through a column of Sephadex G-75 that had been calibrated with markers of known molecular weiglit, and a molecular weight of 16,500 daltons on the basis of sedimentation equilibrium analysis. With p-nitrophenyl phosphate as substrate, the pH optimum was 5.5 and the Michaelis constant was 0.75 mM. The stability of the enzyme at 25° was dependent on pH and... 

Milthorpe BK, Jeffrey PD, Nichol LW. New method for characterizing associating protein systems involving sedimentation equilibrium analysis. P Aust Biophys Chem 1975 3(2) 169—176. 

Philo JS (2000) Sedimentation equilibrium analysis of mixed associations using numerical constraints to impose mass or signal conservation. Methods Enzymol 321 100-120... 

Cassman and King have reported cooperative binding of NADH to beef heart s-MDH (67). They also found evidence for the existence of s-MDH monomer from sedimentation equilibrium analysis at low protein concentrations (67). The fluorescence studies of the binding of NADH to beef heart s-MDH were interpreted in terms of a model in which both monomer and dimer bind NADH with cooperative binding to the dimer (67). The dissociation constant for the first site was about 25 /Jkf and for the second site about 0.18 iiM. 

Essential details of four graft copolymer samples (25a-25d) thus obtained are given in Table 11. The samples have been briefly examined by sedimentation equilibrium analysis. The molecular weights (M ) obtained by this method are consistent with, but lower than, the expected values recorded in Table 11. Another series of amphiphilic graft copolymers with a double comb structure has also been synthesized (26, in Fig. 19) [47]. Similarly, type 27 graft copolymers have been prepared by the reaction of the activated copolymer with hydroxy-terminated polyurethanes, but these have not so far been characterized. 

A second important focus of our work is the development of suitable analytical methods for the solid state and in solution. The physical characterization of metallo-supramolecular systems has mainly relied on crystal structure determination. Studies have also been performed on surface layers 40-42). The classical analytical methods (like FAB mass spectrometry) or most polymer methods (like light scattering, vapor pressure osmometry or membrane osmometry) can not be used. In solution, ESI mass spectrometry (43-45) and NMR (27,46) have been succesfully applied. We have explored whether MALDI-TOF mass spectrometry in the solid state (Schubert, U. S. Lehn, J.-M. Weidl, C. H. Spickermann, J. Goix, L. Rader, J. Mullen, K., unpublished data.) and sedimentation equilibrium analysis in the analytical ultracentrifuge for solutions may be employed. Grid-like cobalt coordination arrays ([2 X 2] Co(n)-Grid) were used as model systems in the analytical ultracentrifuge (47). 

It can be assumed that the corresponding functionalized grids have the same basic structure in view of the similar behavior in UV spectroscopy, the MALDI-TOF mass spectrometry and the preliminary results of sedimentation equilibrium analysis in the analytical ultracentrifuge (48). 

Na-K ATPase is composed of two different subunits, one of about 100000 (a-subunit) and one of about 50000 (yS-subunit), as shown by sodium dodecyl-sulfate (SDS) gel electrophoresis [75,76]. Since both subunits are glycoproteins and therefore bind different amounts of SDS as compared to normal proteins, their SDS gel electrophoretic mobility, and hence their apparent molecular weights can deviate considerably. Recently the molecular weights of the separated subunits of Na-K ATPase from rabbit kidney outer medulla have been determined more accurately by sedimentation equilibrium analysis in the absence of detergents [77]. The value for the a-subunit thus determined is 131000 (120600 for its protein part) and 61 800 for the /8-subunit (42 800 for its protein part). 

The MW of the NADP-specific GDH of Neurospora has been shown to be 280,000-296,000 by sedimentation equilibrium analysis (Wootten, 1973) while the amino acid sequence (Wootten et al., 1974) shows that the polypeptides possess 50 residues less than those of the bovine liver enzyme, and that each subunit has a MW of 48,438 showing that this enzyme also is hexameric. The NADP-specific enzymes of Baker s yeast, E. coli, and the enzyme of dual coenzyme specificity from Mycoplasma laidlawii have been shown to have molecular weights similar to the Neurospora NADP enzyme, while SDS electrophoresis has shown their subunits are also of a similar size (Smith et al., 1975). 

A low molecular weight cellulase (mol. wt. 2.0 x 10 , p/ 7.52) has been isolated from culture filtrates of Trichoderma viride by a two-step procedure of gel filtration and ion-exchange chromatography. Subjection of the preparation to polyacrylamide gel electrophoresis, isoelectric focusing, sedimentation equilibrium analysis, and chromatography of the reduced and alkylated form indicated it to be homogeneous. 

Four electrophoretically distinct endo-(l -> 4)-)3-D-glucanases from Trichoderma viride have been identified. Three of them (mol. wts. 3.72 x 10, 5.20 X 10, and 4.95 x 10 by sedimentation equilibrium analysis) contained D-mannose, o-glucose, D-galactose, and 2-amino-2-deoxy-D-glucose (total 4.5, 15.0, and 15.2 % respectively) and were high in acidic- and hydroxy-amino-acids and glycine, but low in basic amino-acids. 

Human RBP has a molecular weight of 21,200 (Peterson, 1971a) to 21,300 (Raz et al., 1970), as determined by sedimentation equilibrium analysis in the analytical ultracentrifuge. The molecular weight calculated from the reported... 

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. 

Fio. 14. Molecular weight data from high speed sedimentation equilibrium analysis of a-chains ( ) and the fragments of a ( ) and a ( ) resultii from dq don with an ensyme from tadpole. The arrows indicate the average values from several experiments. (Kang el al., 1966b.)... 

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