Stokes radius of the protein

Here rh is the hydrated radius or Stokes radius of the protein. On this assumption s will be expected to increase with the relative molecular mass approximately as Mr2/3. A plot of log s against log Mr should be a straight line. Figure 3-8 shows such a plot for a number of proteins. The plots for nucleic acids, which can often be approximated as rods rather than spheres, fall on a different line from those of proteins. Furthermore, the sedimentation constant falls off more rapidly with increasing molecular mass than it should for spheres. 

A gel filtration (e.g., to Sepharose 6B or CL 6B) yields the Stokes radius of the protein-detergent-phospholipid complex, provided you also run marker proteins of known Stokes radius (e.g., P-galactosidase 6.9 nm apoferritin 6.1 nm catalase 5.2 nm BSA 3.55 nm). The method does not determine the protein MW (because of detergent and phospholipid in the complex). 

Classical gels (Sepharose and Sephacryl) as well as HPLC gels (TSK SW and TSK PW) have been calibrated the elution position, and hence the have been determined for a number of globular proteins of known sizes (refs. 23 and 24). The size of each protein (R) is determined independently by a combination of sedimentation equilibrium and sedimentation velocity experiments so that this parameter is in fact the Stokes radius of the protein (i s) The range which was covered was 20 to... 

Gel filtration is the last of the major chromatography techniques commonly applied in the resolving portion of a process. Of all the techniques discussed thus far in this chapter, gel filtration offers the lowest resolution. The separation is based solely on Stoke s radius of the protein molecule and is the most sensitive to flow rate and sample volume. To achieve significant resolution among sample components, the sample volume should be no greater that five percent of the column bed volume. 

Gel filtration may be used as a means of detecting different conformations of a protein. Kornfeld (137) has found that the elution of Fe3+-saturated transferrin through Sephadex G-100 is retarded relative to the apoprotein. Similar results have been reported by Charlwood (139) who appears to have been able to resolve the three species (O-Fe, 1-Fe and 2-Fe) of transferrin by this method, indicating a 0.7% decrease in the Stokes radius of the molecule per iron atom bound. Differential measurements of sedimentation velocity showed about 1.8% increase in S20W upon binding of 2 iron atoms per mole (139). 

This is also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS). It uses scattered light to measure the rate of diffusion of protein particles in a sample. The data on molecular motion are digitally processed to yield a size distribution of particles in the sample, where the size is given by the mean Stokes radius or hydrodynamic radius of the protein particles this is the effective radius of a particle in its hydrated state. Clearly, the hydrodynamic radius depends on both mass and shape. 

As mentioned earlier, the first signature of the influence of the protein surface on the dynamics of water came from the measurements of the rotational and translational diffusion coefficients of water in aqueous protein solutions. Analysis based on hydrodynamic formulas (such as Stokes-Einstein and Debye-Stokes-Einstein (DSE)) showed that an explanation of the observed values required a larger than actual radius of the protein to be used in the Stokes expression of the friction (from hydrodynamics). This indicated the presence of a substantially rigid water layer around the protein surface. However, the story turned out to be more complex. We have already discussed some of these aspects - we now turn to a more detailed discussion of several experimental results. 

We determined the Stokes radius of the second peak, by calibration of the column with standard proteins and also by another technique of equilibrium saturation gel filtration which proved very... 

Rigid, unsolvated spheres. Stokes law, Eq. (9.5), provides a relationship between f and the radius of the particle. Since this structure is a reasonable model for some protein molecules, experimental D values can be interpreted, via f, to yield values of R for such systems. Note that this application can also yield a value for M, since M = N pj [(4/3)ttR ], where pj is the density of the unsolvated material. 

Stokes radius determination. The Stokes radii (Rs) of DnaK, RCMLA, NCA-SNase and their complexes were determined by SEC-HPLC using a series of standard globular proteins for which Stokes radii were available (15. and references therein). It is well established that size-exclusion partition coefficients can be correlated to the moleculzir mass, MM, of proteins by eq. 3 ... 

However, surfactants incorporated into the electrolyte solution at concentrations below their critical micelle concentration (CMC) may act as hydrophobic selectors to modulate the electrophoretic selectivity of hydrophobic peptides and proteins. The binding of ionic or zwitterionic surfactant molecules to peptides and proteins alters both the hydrodynamic (Stokes) radius and the effective charges of these analytes. This causes a variation in the electrophoretic mobility, which is directly proportional to the effective charge and inversely proportional to the Stokes radius. Variations of the charge-to-hydrodynamic radius ratios are also induced by the binding of nonionic surfactants to peptide or protein molecules. The binding of the surfactant molecules to peptides and proteins may vary with the surfactant species and its concentration, and it is influenced by the experimental conditions such as pH, ionic strength, and temperature of the electrolyte solution. Surfactants may bind to samples, either to the... 

Protein-Losing Syndromes (in Association with Hp 2-i or 2-2 Phenotypes). Most protein-losing syndromes, such as the nephrotic syndrome, are associated with sieving loss of plasma proteins, with excretion inversely proportional to the effective diameter of the protein (Stokes radius). There is a concomitant, compensatory increase in synthesis of most if not all proteins synthesized in the liver, including Hp. Hp 1-1 is relatively small and is lost in approximately the same proportion as albumin. However, the 2-1 and 2-2 variants are large these are retained, and elevated concentrations are usually seen unless hemolysis is also present. 

Membrane inactivation depends on how closely anions can approach cationic binding sites. Poorly solvated ions show the strongest binding. They are also known to be the most effective protein denaturants (65). The Stokes law hydrated radius of the toxic bromide anion is about 1.2 A, that of the relatively nontoxic fluoride about 1.6 A and that of the cryoprotective acetate anion 2.2 A. Biological membranes usually appear in thin sections as three-layered structures 60 to 100 A thick. In view of this relatively large cross section, accessibility of binding sites becomes of obvious importance. 

The deoxycholate-solubilized squalene synthetase was also chromatographed on Sephadex G-200 and a Stokes radius of 40 A was found for the enzyme. Again, the two catalytic activities were not resolved [72]. This value, along with the 20,w indicated a molecular weight of 55 000 for the protein(s). This contrasts with much higher values reported earlier. Inclusion of certain phospholipids in the tubes used... 

Fig. 2. The Stokes radius of d"b5monomer as determined by both small zone and equilibrium saturation gel filtration. The five standard proteins used to calibrate the small zone ( - - - ) and...

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