Sedimentation-velocity technique

Sedimentation equilibrium technique, 19 256 Sedimentation velocity technique, 19 256 Sediments, arsenic in marine samples, 44 149, 162-164, 169, 181 [Se U ] cations, 35 297-298 Selective vaporization, for preparation of actinide metals, 31 12-13, 26 Selenide, production, 38 82 Selenium... 

This sedimentation-velocity technique readily separates proteins differing in sedimentation coefficient by a factor of two or more. 

The dissociation of apoferritin at extremes of pH was examined by sedimentation velocity techniques (166). It was established that between pH 2.8—10.6 the apoferritin monomer ( 17S) was the only species that could be detected. Between pH 2.8—1.6 and 10.6—13.0 both monomer (17S) and a low molecular weight component (2—3S), presumed to be subunit, were detected. The dissociation follows a smooth sigmoidal curve (Fig. 7 a) in both cases with mid points, corresponding to equal amounts of 17 S and 2—3S component at 2.2—2.4 for the acid dissociation and 11.8—12.2 for the alkaline dissociation (the exact values are dependent on the buffer used). When apoferritin is completely dissociated into subunits at low pH (either by exposure to buffer of pH 1.6, or by treatment with 67% acetic acid) and is then dialysed into buffer of higher pH, reassociation does not take place in dilute glycine buffers until pH values in excess of that required to induce subunit dissociation (Fig. 7 a). The reassociation then follows a sigmoidal curve until complete reassociation to a 17S monomer is attained at pH 4.3. Although we have not as yet been able to follow reassociation completely in a more concentrated buffer (200 mM cf 10 mM) it is clear (Fig. 7 a) that subunit reassociation occurs at a much lower value and that the hysteresis observed between dissociation and reassociation is much less in the more concentrated... 

A particular advantage of the sedimentation-velocity technique is that a macro-molecular solution containing more than one type of molecule is separated according to the molecular masses of the components. Figure 11.156 shows the type of sedimentation diagram obtained for a system containing a number of components. 

Weight-average molecular weight is determined by Light Scattering and Ultracentrifugc/Sedimentation-velocity techniques which depend mainly on the size/weight of the polymer molecules and only to a small extent on their number. 

Partial etherification of the beech wood MGX with p-carboxybenzyl bromide in aqueous alkali yielded fully water-soluble xylan ethers with DS up to 0.25 without significant depolymerization the Mw determined by sedimentation velocity was 27 000 g/mol [400,401]. By combination of endo- 6-xylanase digestion and various ID- and 2D-NMR techniques, the distribution of the substituents was suggested to be blockwise rather than uniform. The derivatives exhibited remarkable emulsifying and protein foam-stabilizing activi-... 

Diffusion coefficients can also be obtained from sedimentation velocity experiments, but the precision is quite low and subject to some ques-don. Quasi-elastic light scattering is a much more useful technique to obtain diffusion coefficients (see below). 

Indirect methods for obtaining information on the kinetics of the associa-tion/dissociation equilibrium include sedimentation velocity and GPC experiments. The application of these techniques is based on comparison of sedimentation or GPC elution curves with model curves based on theories for separation of unimers and micelles during a sedimentation velocity (Gilbert 1955) or GPC (Ackers and Thompson 1965 Coll 1971 Prochazka et at. 1988, 1989) experiment. Experiments have been performed that demonstrate several of the qualitative model predictions (Prochazka et at. 1989). The main conclusions were that GPC curves with two well-separated peaks can only result from a slow dynamic molecule micelle equilibrium, and that no simple interpretation of elution curves in terms of relative concentrations of unimer and micelles is possible (Prochazka et at. 1989). Thus no quantitative information on the kinetics of the molecule micelle equilibrium can be obtained from sedimentation velocity or GPC data. 

Relative techniques include gel permeation chromatography (GPC) (Anger and Berth, 1986 Deckers etal., 1986 Berth and Lexow, 1991 Hourdet and Muller, 1991), sedimentation velocity and viscometry (Christensen, 1953). These methods require assumptions of molecular conformation or calibration using standards of known MW. 

When used properly, ultracentrifugation continues to be our major means of determining molecular weight values for humic substances using sedimentation velocity and other techniques. Indeed, centrifugation studies with humic substances have usually centered upon molecular weight measurements. 

The reversible dissociation and reassociation has also been studied at low pH by gel filtration, and it is clear from these results (767) that during both dissociation and reassociation both subunit dimers and subunit tetramers were observed in addition to monomer and subunit. The ratio of monomer to subunit + subunit dimer + subunit tetramer observed by gel chromatography were quite different to that found by sedimentation techniques this is illlustrated for subunit dissociation in Fig. 8, where at pH 2.16 the ratio of fast/slow component in sedimentation velocity is 1 1, the ratio of monomer to subunit -f subunit oligomers is 1 20. In contrast, the ratio of monomer to subunit + subunit oligomers at pH 3.8 is 20 1 from gel filtration and 1 1 by sedimentation velocity. The imposition of a slow transport process on the equilibrium in gel filtration may affect the equilibrium be mass action so that rate limiting steps are affected. On this assumption we might assume that the rate... 

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