Protein sedimentation constants

Perhaps the strongest evidence in favor of the theory that the cytochrome oxidase system is composed of a single protein comes from the physicochemical studies of Takemori. Purified cytochrome oxidase appears in the ultracentrifuge as a nondispersed protein (sedimentation constant = 21.9 diffusion constant =3.58 x 10 partial specific volume = 0.72). The molecular weight of the protein was calculated on the basis of these studies and was found to equal 530,000. 

Once the amino acid has been bound to its tRNA, it can pass to the next phase of protein synthesis, involving its interaction with mRNA, which takes place on the ribosome, a molecular machine of enormous complexity. The ribosome of E. coli is a ribonucleoprotein assembly of molecular weight 2700 kDa, and sedimentation constant of 70S9. It is made up of roughly two-thirds RNA and one-third protein, and can be separated into a small (30S) and a large (50S) subunit. The 30S subunit contains 21 proteins and one 16S RNA molecule, while the large subunit has 34 different proteins and two RNA molecules, one 23S and one 5S. Despite its size and complexity, the structure of both ribosomal subunits has been determined to atomic resolution (Figure 4.32), and very recently the atomic structure of the 70S ribosome has been determined at 2.8 A resolution (Selmer et al., 2006). 

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. 

From analysis of a variety of well-characterized proteins, Squire and Himmel192 observed that if proteins are assumed to contain 0.53 g H20 per gram of protein and to have a mean value for v of 0.730 g / cm3 the value of Mr can be predicted by Eq. 3-17 with the standard deviation indicated. Here, S is the sedimentation constant in Svedberg irnits (10 13s). 

Table 5-4. There are many others.302 In addition, there are thousands of different mRNAs within a cell. The most abundant RNA molecules in cells are those of the ribosomes. Ribosomes consist of two elaborate RNA-protein subunits, a large subunit with sedimentation constant 30S in bacteria and 40S in eukaryotes.

Fig. 1. Effect of pH on the corrected sedimentation constant of conalbumin at ionic strength 0.1, protein concentration 1.1—1.3 g./lOO ml. , conalbumin prepared by electrophoresis—convection O. conalbumin prepared by the acid precipitation method Q, conalbumin prepared by electrophoresis—convection, exposed for 1 hr. to pH 2—3 before sedimentation at pH 5.2 A, conalbumin prepared by the acid precipitation method, exposed for 1 hr. to pH 2.5—3.4 before sedimentation at pH 4.5—7.5. (Arch. Biochem. Biophys. 61, 51 [1956]).

The purified testis protein as prepared maintained a single homogeneous boundary in ultracentrifugation. The sedimentation constant (S20, w) was calculated to be 1.9 S on the assumption of a partial specific volume of 0.72. 

The iron content of testis non-heme iron protein was found to be 103 mji, atoms per mg of protein on a biuret basis. Although the sedimentation constant is slightly higher than that of adrenodoxin, it would be reasonable to assume that there are 2 iron atoms per mole of protein. 

Racemization rates (Table I) clearly differ among these seven amino acids. To compare results from the four proteins, rate constants were calculated from these data. For casein, D/L ratios were measured at 0, 1, 3, 8 and 24 hours. These results are plotted in Figure 2. The curves have two regions of different racemization rates. Rapid initial rates observable up to about 3 hours are followed by slower rates up to 24 hours. The amino acids apparently have not reached equilibrium by 24 hours of incubation. Theoretically for amino acids having one asymmetric center, the equilibrium D/L ratio is 1.0. This value has been observed in fossil bone protein (see 13) and in dry roasted proteins (10), but not in calcareous marine sediments (17) nor in fossil mollusc shell (18). 

The formation of polymer-polymer complexes as a rule is observed in aqueous media5,33. The viscosity of complexes in water is about 0.05-0.10 dl/g and close to that of globular proteins. Aqueous solutions have some features low intrinsic viscosity values are independent of the matrix molecular weight, the absence both of the concentration dependence of the reduced viscosity and the polyelectrolyte anomaly, and high values (about 30 s) of the sedimentation constant. 

The molecular weight of cytochrome c peroxidase has been determined to be 34,100 on the basis of a sedimentation constant of 3.55 S, a diffusion constant of 9.44 F, and a partial specific volume of 0.733 ml/g (4 )-The enzyme exists as a monodisperse monomer containing one ferric protoporphyrin IX, which is noncovalently bound (/, , 14). No other transition metal is detected in crystalline preparations of the enzyme (22). The apoenzyme moiety is an acidic protein with an isoelectric point at pH... 

The molecular-sieving effect on proteins has been very clearly demonstrated by Pedersen (1962). Bovine serum albumin, obtained by fractionated ammonium sulfate precipitation, was filtered through a column of cross-linked dextran of water regain 15 gm/gm (Sephadex G-200). Monomer and dimer forms were easily separated from each other and from other components of larger sedimentation constants (Fig. 8). Heterogeneity in the monomer as well as in the dimer preparation was indicated... 

The anal5 sis of Tanford et al. which covers twelve different proteins, shows that the following equation for the intrinsic viscosity and sedimentation constant at 25° in the denaturing solvent is valid for all the proteins considered ... 

The determination of frictional ratios from measurements of sedimentation constant or diffusion constant or both, for proteins of known molecular weight. 

If the molecular weight of a protein is known from measurements of osmotic pressure, of sedimentation and diffusion, of sedimentation equilibrium, of light scattering, or by any other inethod, and if its partial specific volume is also known, then the frictional ratio fjf may be determined either from its sedimentation constant or its diffusion constant. The frictional coefficient / is that characteristic of a spherical unhydrated molecule of the same molecular weight and partial specific volume as the protein under consideration. If r is the radius of tliis hypothetical sphere and rj the viscosity of the medium, then... 

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