Low-speed sedimentation equilibrium

Information on polydispersity of PDADMAC samples synthesized under different conditions could be obtained further by the Mz/Mw -ratio from low-speed sedimentation equilibrium experiments in 0.5 m NaCl [35]. 

It should also be pointed out that light-scattering may be the only absolute method applicable to species having molecular weights larger than 15 X 10 (the upper limit for accurate measurements from low-speed sedimentation equilibrium procedures, unless ultra-short solution columns are used). 

Despite these difficulties, and only after the employment of meticulous preparative procedures, reproducible results were obtained by Carlstedt and Sheehan for T-domains, subunits, and whole mucins, which appear to be in agreement with other data. For whole pig-gastric mucin, for example, they obtained a molecular weight of 45 X 10 , some 20 times the value reported by Allen and coworkers. Lower values (15-40 X 10 ) have also been obtained, - presumably because of sample variability. Using low-speed sedimentation equilibrium, Creeth and Cooper obtained a lower value ( 9 X 10 ). 

Fig. 5.—Rayleigh Interference F rofiles from Low-speed Sedimentation Equilibrium, ((a) Native mucin (bronchial gp 376) (b) reduced gp 376 (c) immunoglobulin G. In (a), note the steep rise of the fringes near the cell base, but the finite slope at the meniscus.]...

The plot of log (concentration) vs. (radius)2, obtained during both low- and high-speed sedimentation equilibrium analyses, was invariably linear across all portions of the solution column which could be examined (13, 16, 17). 

In a sedimentation equilibrium experiment the cell is rotated at a relatively low speed (typically 5000-10000 rpm) until an equilibrium is attained whereby the centrifugal force just balances the tendency of the molecules to diffuse back against the concentration gradient developed. Measurements are made of the equilibrium concentration profiles for a series of solutions with different initial polymer concentrations so that the results can be extrapolated to c = 0. A rigorous thermodynamic treatment is possible and enables absolute values ot Mwand Mz, to be determined. The principal restriction to the use of sedimentation equilibrium measurements is the long time required to reach equilibrium, since this is at least a few hours and more usually is a few days. 

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 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 without denaturing the protein. Thus the native quaternary structure of multimeric proteins is preserved In contrast, SDS-polyacrylamide gel electrophoresis... 

The boundary is a concentration gradient, and solutes will tend to diffuse against the gradient. At low centrifugal speeds, diffusional spreading will oppose sedimentation until eventually an equilibrium is achieved. Sedimentation equilibrium experiments lead to static exponential concentration distributions like the one shown in Figure 2. 

Sedimentation equilibrium A technique for using centrifugation to measure the mass of a large molecule such as a protein. A solution of the substance is centrifuged at low speed until the tendency of the substance to sediment is balanced by its tendency to diffuse to uniform concentration the resulting concentration gradient is used to measure the molecular mass. 

Centrifugation Incomplete sedimentation, low speed, short time. Complete sedimentation till equilibrium is achelved, high speed, long time. 

The sedimentation equilibrium method, in which diffusion plays an equally important role. This is done at a relatively low speed, for example, 12,000 rpm. 

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