Partial specific volume, determination

Lovenberg, Buchanan, and Rabinowitz 65) showed that the molecular weight of C. pasteurianum ferredoxin is about 6000, based on sedimentation velocity and sedimentation equilibrium ultracentrifugation determinations and on amino acid analysis. The sedimentation coefficient, S2o,w was 1.4, and the partial specific volume, determined according to the method of Hvidt et al. 59) was 0.63, as compared to the value of 0.71 observed for most proteins. Similar investigations showed that ferredoxins from four other clostridia Lovenberg, Buchanan, and Rabinowitz 65)) and from a photosynthetic bacterium (Bachofen and Arnon 12)) also had a molecular weight of about 6000. 

Table 3 Refractive index increment and partial specific volume of hyaluronan (M 10 g/mol) determined at different salt concentrations (from [28])...

The amount of hydrated biopolymer and of free water in the biopolymer-water system, the thermodynamic notion of partial specific volume has been introduced and is frequently determined. The relation to Vsp, the specific volume, is shown by the equation ... 

The viscosity increment was determined as v = B v = 172.7 ( 2 5 for spheres) where B is the viscosity coefficient characteristic of a given solue-solvent pair, and amounts to (9.91 0.24)xl0" ff/ A g" for PGA in aqueous solution. is the partial specific volume of the macromolecular component equal to [Pg.612]

Sedimentation- diffusion Absolute fairly fast Requires 3 separate determinations (diffusion coefficient, sedimentation constant and partial specific volume) <5... 

The diameter d of a polymer chain can be estimated from (1) hydrodynamic quantities such as intrinsic viscosity and sedimentation coefficient, (2) the partial specific volume vgp of the polymer, and (3) X-ray crystallographic data of the polymer. Table 2 lists the values of d for liquid-crystalline polymers estimated by different methods. Those determined from hydrodynamic data are close to but slightly larger than those from vsp and crystallographic data, though this may not always be the case. 

Sedimentation. The sedimentation experiments are tabulated in Tables I and II. In Table I typical sedimentation coefficients determined in H20 and D20 are in close agreement here and with previously reported values determined for both protio and deuterio phycocyanin from F. calothricoides (15,16). Each of the tabulated coefficients is for a single experiment at an approximate protein concentration of 15 mg. per ml. Lyophilizing a phycocyanin preparation twice had little effect on the observed sedimentation coefficients. In calculating the S values the same partial specific volume of the protein was used for both D20 and H20. This practice is consistent with the recent results of Edelstein and Schach-man (7). Small increases in sedimentation coefficients from H20 to D20 are to be expected because of deuterium substitution on exchangeable positions. The slope of an S vs. concentration plot for phycocyanin in H20 and D20 would also probably differ. Consequently, small changes in S from H20 to D20 would be expected at a constant protein concentration. 

Lactoglobulin A in 40% 2-Chloroethanol. Previous light scattering and differential refractometry measurements (8, 23) have shown that / -lactoglobulin exhibits strong preferential interactions with solvent components in the water-2-chloroethanol system. Since the preferential interaction between protein and 2-chloroethanol in this system was found to be maximal at 40% (v/v), the effect of this interaction on the partial specific volume of the protein was determined. 

The densities of the solvent and of / -lactoglobulin A ( -Lg) in 40% (v/v) 2-chloroethanol, in the presence of 0.01 M HC1 and 0.02M NaCl, were determined, with and without prior dialysis, in a 10-ml pycnometer at 20°C. Solutions were prepared as described previously (8, 24). The solutions were filtered through millipore filters in syringe adapters just before the density measurements. Protein concentrations were determined after filtration by ultraviolet absorption at 278 nm. The apparent partial specific volume, oapp, was calculated from the densities using the standard equation (21, 25) ... 

Knowledge of the partial specific volume of a protein in the denaturant, t>2,03, which is essential for determining its molecular weight by small-angle x-ray scattering, permits also the calculation of the volume change upon denaturation since... 

For the analysis of light scattering experiments the refractive indices of the DADMAC/AAM solutions at dialysis equilibrium were determined, showing the validity of the additivity principle [67]. The additivity could also be proven for the partial specific volumes which are necessary to calculate molar masses from ultracentrifugation experiments [131]. These dependencies are summarized in Fig. 24. 

A value of 2.5 was assigned to the viscosity increment, v, assuming micellar sphericity. The partial specific volume, v, was determined from density measurements. 

Pure adrenodoxin can be obtained by either procedure A or B when the steps of DEAE-cellulose chromatography and Sephadex G-75 passage are repeated. Fig. 1 shows data of the sedimentation equilibrium experiment the result indicates that the molecular weight is approximately 12,000. From sedimentation velocity experiments, the sedimentation constant (Szo.w) was calculated to be 1.55 S. The diffusion constant (Z)2o, w) measured in a Neurath cell was computed to be 11 x 10-7 cm2/sec. The determination of partial specific volume by measurement of the... 

M is the weight of sodium bromide to be added to a volume v of solution to change its density from d to d2 (at a stated temperature) and v is the partial specific volume of the salt at the relevant temperature and concentration. Values of v can be determined for sodium bromide applying the data of Baxter and Wallace (see Mills et al.). 

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... 

Fig. 5. Hydration dependence of the volume of human serum albumin crystals. Specific volume of crystals of the dimer form of human serum albumin as a function of water content, o, Determined during drying , determined during rehydration. Extrapolated intercepts of solid line Up = 0.734 cm /g and = 0.995 cm= /g. The dashed line with arrowheads indicates the region of failure of the simple linear relationship and of deviation of partial specific volumes of protein and water from the dilute solution values. From Richards (1977).

Values kindly determined by Mr. D. M. Brown and Prof. E. L. Smith on a sample prepared in this laboratory. The values have been obtained, respectively, by the Archibald method of sedimentation equilibrium and by sedimentation-diffusion, assuming a partial specific volume of 0.721 for the protein. 

Here M is the molecular weight and V the partial specific volume of the solute, N the Avogadro number, k the Boltzmann constant, and T the absolute temperature s and D are the sedimentation and translational diffusion coefficients (after extrapolation to infinite dilution). The translational frictional coefficients from both measurements are regarded as identical, i.e., f, = fd. The rotary frictional coefficient, designated as f, can be determined from either flow birefringence or non-Newtonian viscosity measurements. 

For molecular modeling of the lipoproteins, values for the partial specific volumes of the lipoprotein components are required. The partial specific volume of an aqueous egg yolk lecithin suspension is 0.984 ml/g (Hauser and Irons, 1972), and this provides a reasonable approximation for the partial specific volume of the phospholipid occupying the surface monolayer of a lipoprotein. The reciprocal of the density of liquid triolein (Small, 1986) yields its partial specific volume, 1.102 ml/g, and provides a reasonable approximation for triglyceride dissolved in the cholesteryl ester-filled core of the LDL. For cholesterol, the partial specific volume of 1.021 ml/g measured in benzene (Haberland and Reynolds, 1973) has been employed. The value of 0.740 ml/g employed for the partial specific volume of apoBlOO was determined from its amino acid composition (Lee et al., 1987). A value of 0.60 ml/g was used for the partial specific volume of the carbohydrate moiety. One important parameter, the partial specific volume of cholesteryl ester, remains to be determined. As will be shown below, its value is estimated to be 1.058 ml/g. 

Lundberg. Bull s data at T = 298.15 K have been used for the activity of water in this system." Starkweather has determined the activity of water in the concentration range 0 volume fraction as ai = 12 1, the expression that was used in our calculations. The molecular weight and the partial specific volume of collagen were taken from ref 41. The results of the calculations are presented in Figures IB, 2B, and 3B. In contrast to the toluene + polystyrene mixture, the solvent (water) is in deficit around a central water molecule but in excess around a protein molecule. 

The primary technical limitation on the accuracy of molecular weights so determined is the sedimentation constant. Since the molecular weight is directly proportional to the sedimentation constant, where S is low, small errors in S will be relatively important. For -dextrin with S = 0.47, if S is in error by only 0.02 (an average error for measurements of this sort) this will give an error of 50 in the molecular weight. A second essential factor which so far has not been determined with high precision is the partial specific volume of the carbohydrate. Nevertheless, it has been possible to obtain molecular weights which are within a few perct nt of the theoretical values (see Table IV). 

The D-xylanase system of Stereum sanguinolentum,199 the only D-xylanase for which an amino acid composition has as yet been published, was found to contain a high proportion of acidic and aromatic amino acid residues. The M.W., as determined from the amino acid composition, is 23,900, compared with 21,600 as calculated from ultracentrifugation data. Other physical parameters that have been determined199 for this D-xylanase include the sedimentation coefficient [2.8S, which is similar to that reported for a D-xylanase isolated from Trichoderma viride,203 namely, 2.IS], the partial specific volume (0.71 cm3.g 1), and the molar extinction coefficient (6.25 X 104). Activation energies have been reported for D-xylanases from Schizophyllum commune233 (EA 28.6 kj.mol-1) and from a commercial cellulase preparation229 (EA 34.0 kj.mol-1). 

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... 

---