Sedimentation coefficient apparent

In a solution of molecules of uniform molecular weight, all particles settle with the same value of v. If diffusion is ignored, a sharp boundary forms between the top portion of the cell, which has been swept free of solute, and the bottom, which still contains solute. Figure 9.13a shows schematically how the concentration profile varies with time under these conditions. It is apparent that the Schlieren optical system described in the last section is ideally suited for measuring the displacement of this boundary with time. Since the velocity of the boundary and that of the particles are the same, the sedimentation coefficient is readily measured. 

Ribosomes are ancient ribonucleoprotein complexes that are the sites of protein synthesis in living cells. Their core structures and fundamental functional mechanisms have been conserved throughout the three domains of life bacteria, archaea and eukaryotes. All ribosomes are organized into two subunits that are defined by their apparent sedimentation coefficient, measured in Svedberg units (S). There is a general... 

The simplest way computationally of obtaining a sedimentation coefficient distribution is from time derivative analysis of the evolving concentration distribution profile across the cell [40,41]. The time derivative at each radial position r is d c r,t)/co /dt)r where cq is the initial loading concentration. Assuming that a sufficiently small time integral of scans are chosen so that Ac r t)/At= dc r t)ldt the apparent weight fraction distribution function g (s) n.b. sometimes written as (s ) can be calculated... 

A sedimentation coefficient distribution—either c s) versus 5 or g (s) vs. s—for a polysaccharide can also be converted into an apparent molecular weight distribution if the conformation of the polysaccharide is known or can... 

The thus determined molecular weight is an apparent one since s and D depend on the polymer concentration. Therefore, extrapolation to concentration zero is required. The sedimentation coefficient obtained by extrapolating c —> 0 is called sedimentation constant Sn ... 

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. 

Although hydrolyzed iron (III) solutions are unstable with respect to eventual precipitation of hydroxides (sec. II A 3), Feitknecht and Mi-chaelis (29) observed no correlation between the time required for precipitate formation and the degree of hydrolysis. Furthermore, while on standing the polymeric fraction in hydrolyzed ferric nitrate solutions increases slowly in sedimentation coefficient, this process is not parallel to precipitate formation (37). Apparently the polymer is not an intermediate in precipitate formation. 

The apparent molecular weight of asparaginase in 8M urea, 5M guanidinium chloride, or 9Af formamide was first reported as being 19,000-24,000, with an s°o.w of 1.5 to 1.8 S (77), demonstrating the presence of subunits. Arens et al. (65) and Frank and Veros (78) have confirmed the value of the sedimentation coefficient of the subunits obtained in 8 M urea, but the latter authors reported an apparent molecular weight of approximately 32,500 instead of 19,000-24,000, and their value is almost certainly the correct one. 

Fig. 3.29 Dependence of (a) apparent molecular weight, (b) sedimentation coefficient and (c) intrinsic viscosity fi/, and Huggins coefficient, ktl. on solvent composition (methanol-water-0.1 M LiBr mixtures) for solutions of a PS-poly(4-vinyl-A -ethylpyri-dinium bromide) = 32.7kgmol l, 8% PS) diblock (Selb and Gallot 1981a).

It is recommended to carry out a set of SV measurements of the unfolded and the folded state ensembles as a function of RNA concentration as described earlier before collecting the data points for the entire equilibrium transition. The shape of the apparent sedimentation coefficient distribution curves (discussed in Section 5) derived from such measurements, indicate whether the initial and the final states are conformationally homogeneous. Appearance of more than one peak in such distributions would indicate the existence of distinct structural states with different hydrodynamic properties. 

The radial concentration scans obtained from the UV spectrophotometer of the analytical ultracentrifuge can be either converted to a radial derivative of the concentrations at a given instant of time (dc/dr)t or to the time derivative of the concentrations at fixed radial position (dc/dt)r (Stafford, 1992). The dcf dt method, as the name implies, uses the temporal derivative which results in elimination of time independent (random) sources of noise in the data, thereby greatly increasing the precision of sedimentation boundary analysis (Stafford, 1992). Numerically, this process is implemented by subtracting pairs of radial concentration scans obtained at uniformly and closely spaced time intervals c2 — G)/( 2 — h)]. The values are then plotted as a function of radius to obtain (dc/dt) f versus r curves (Stafford, 1994). It can be shown that the apparent sedimentation coefficient s ... 

Fig. 10.2B). At this stage, you may also convert the apparent sedimentation coefficient 5 to the standard apparent sedimentation coefficient 5 0 w ... 

The apparent sedimentation coefficient distribution function, g(s ) vs. s, can be computed from the time derivative of the concentration profile using the following equation, in which the time derivative of the concentration profile has been corrected for the "plateau" contribution by an iterative procedure described previously (Stafford, 1992a Stafford, 1994a) ... 

Stafford, W. F. (1994b). "Sedimentation Boundary Analysis of Interacting Systems Use of the Apparent Sedimentation Coefficient Distribution Function" in MODERN ANALYTICAL ULTRACENTRIFUGATION Acquisition and Interpretation of Data for Biological and Synthetic Polymer Systems Eds. T. M. Schuster and T. M. Laue. Boston, Birkhauser Boston, Inc. 

An extended analysis of data using the time-derivative method provides for simultaneous determination of apparent sedimentation, and apparent diffusion coefficient, values at a particular concentration and temperature [9]. The apparent diffusion coefficient was calculated from the apparent sedimentation coefficient distribution by the following relationship ... 

Figure 4. Sedimentation Velocity Analysis of ZDD. A, Primary data collected at 1 mg/ml (10 scans). B, Apparent sedimentation coefficient distribution function, g(s ) versus s. The error bars represent the standard error of the mean. The solid line is the fit to equation 4. Apparent s, D, and Ms,D values were calculated as described.

In the two different membrane domains FPR were expected to show different coupling to G proteins. Solubilized FPR prepared from the PM-L fraction of neutrophils sedimented in a detergent-containing sucrose density gradient as 7 S particles while FPR prepared from PM-H sedimented with an apparent sedimentation coefficient of 4 S. The 7 S form was shifted to the 4 S form in a GTPyS-sensitive manner indicating that the 7 S form represents receptor-G protein complexes. 

Many physical characteristics have been determined for the mushroom tyrosinases. These include diffusion coefficients, sedimentation coefficients, frictional ratios, Stoke s radii, isoelectric points, and apparent molecular weights (17-20). Recent evidence suggests that tyrosinase is composed of two heavy chain components (H) of approximately 43-45 kd each and two light chain components (L) of approximately 13 kd each (18-20). The heavy chains contain the catalytic sites. Podila and Flurkey (21) have suggested that the heavy chains were synthesized as smaller molecular weight precursors to the native enzyme. Different isoenzyme forms of tyrosinase apparently contain different H chains (7,17). No role for the L chain subunits have been reported. 

An additional example of how enzymes can affect the physical properties of purified proteins relates to the enzymic dephosphorylation of /3-casein (4). Bovine /3-casein contains five phosphate groups per monomer as phosphoseryl residues. Purified /3-casein from bovine milk was dephosphorylated by a phosphoprotein phosphatase. Both the native /3-casein and 65% dephosphorylated /3-casein self-associated to form polymers at 35°C. However, the dephosphorylated /3-casein had a larger sedimentation coefficient (S35 22.5) than that of the native /3-casein (S35 18.2). Also the sedimentation pattern of the dephosphorylated /3-casein was more polydisperse than the hypersharp pattern of the native /3-casein. These properties were accentuated with 95% dephosphorylated /3-casein. The decrease in the negative charge caused by the loss of phosphate apparently favors the self-association of /3-casein resulting from hydrophobic bonding. 

From solubilized membrane proteins you can— without major equipment—determine MW, Stokes radius, sedimentation coefficient, proportion of bound detergent and phospholipid, isoelectric point, and the apparent frictional coefficient. Knowledge of Stokes radius, MW, and isoelectric point comes in handy during planning of a purification or drafting of a detection assay (the other measures are good for the library). 

This study led to the remarkable conclusion that the allosteric regulation of class II enzymes responds to the same rules as class I, described above. However, some of the class II RNRs, for example the one from L. leichmannii or that from T. maritima, contain only the specificity site, as in class Ib. This site has a very high affinity for nucleotides with a Kj value for dATP of 15 nM for the thermophilic RNR. The allosteric effectors seem to decrease the apparent K value for the appropriate substrate and the K value for AdoCbl. Many lines of evidence indicate that the binding of allosteric effectors induces a considerable conformational change, as shown from changes of the CD spectrum and of the sedimentation coefficients, quenching of protein fluorescence, and changes of the EPR spectrum of protein-bound cob(n) alamin. 

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