Sedimentation velocity molecular weight determination

Diffusion coefficients can be related to molecular weight in three ways first by application of the Stokes-Einstein equation, second by combination with sedimentation data, and third by consideration of homologous polymer solutions. In the first method, an equivalent spherical size of the molecules is calculated from Dt, and an approximate molecular weight is found by combining these data with the appropriate density. In the second method, diffusion measurements are coupled with those of sedimentation velocity to give molecular weights, and in the third method, molecular weights may be determined directly from measurements of diffusion coefficients alone once a calibration has been... 

To perform this verification three narrow fractions of linear polystyrene denoted as A, B, and C were investigated to determine their molecular weights. This task was accomplished by applying velocity as well as equilibrium sedimentation. In addition, these three individual fractions were combined according to a known weight ratio into a new polydisperse sample denoted as P. Sample P was also subjected to equilibrium sedimentation. All samples were investigated in cyclohexane at 35°C (near the 0 temperature) see Table I. 

The molecular weight of the calf duodenal adenosine aminohydrolase determined from sedimentation and diffusion data (91) and by comparative elution from Sephadex gels (93, 94) ranges from 31,000-35,000 (84,91, 93, 95). A molecular weight of 52,000 from sedimentation velocity and sedimentation equilibrium data has not been confirmed (93). 

Some of the physicochemical parameters of the enzyme, as determined on the Kunitz preparation (S), are summarized in Table I. Sedimentation equilibrium analysis of molecular weight carried out on the enzyme crystallized from ammonium sulfate gave a value of 71,000 (5), in fair agreement with the value of 63,000 obtained by sedimentation velocity measurements on the Kunitz preparation (10). The enzyme has been reported to dissociate into subunits in the presence of sodium dodecyl... 

The ultracentrifuge can be used to separate fractions of different molecular weights in a mixture of solutes and to determine the molecular weight of a solute. Two different approaches can be taken. In the sedimentation velocity approach, the change in the concentration profile with time is determined during the centrifugation process. This nonequilibrium process requires a knowledge of diffusion rates and is not based directly on thermodynamics. We will leave a discussion of this process to other texts. 

The molecular weight is an intrinsic property of biomolecules. Sedimentation velocity is one of physicochemical methods which have been employed to determine molecular weights of many biomacromolecules. The molecular weight (M) of a biomacromolecule can be computed according to the Svelberg equation ... 

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. 

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... 

The purified hemagglutinin was homogeneous by gel filtration and immuno-electrophoresis, and in the analytical ultracentrifuge.63,560,561 A molecular weight of 79,000 was determined by the sedimentation equilibrium method561,569 (100,000 by sedimentation and velocity measurements,83 and 53,000 by gel filtration563). 

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. 

Price and Radda (338) found that N-acetylimidazole could acetylate up to six tyrosine residues without loss of activity or alteration of Km for substrate however, reaction of about one tyrosine per subunit results in desensitization toward GTP, but the response to ADP is not abolished even by extensive 0-acetylation. Essentially the same results are observed upon nitration with tetranitromethane (TNM). Acetylation does not grossly alter the molecular weight, as measured by sedimentation velocity, or the conformation, as determined by ORD. The GTP site is not protected by NADH alone, but is partially protected (25-50%) by GTP and is at least 75% protected by inclusion of both GTP and NADH in the reaction mixture. Piszkiewicz et al. (339) confirmed these findings by modification with TNM. The reaction is biphasic with initial rapid formation of one residue of 3-nitrotyrosine per subunit. The primary site of reaction is tyrosine-406 in the linear sequence (340). Later (338) the same effect was obtained with chicken GDH with both enzymes there is no influence on activation by ADP. Further, the pH optima of the enzymes are not influenced by the degree of nitration or the inhibition by GTP or activation by ADP (338). 

A molecular weight of 76,600, determined by sedimentation efficient measurements, was used in calculation. velocity-diffusion co-... 

Sedimentation velocities of DNAs can be determined as described by Prunell and Ber-nardi (1973), and molecular weights then calculated from the sedimentation coefficients 20,w, using the relationship of Eigner and Doty (1965)... 

Other properties of algin reported include conductivity (252 253), contact angles of various solvents on different metal alginate films 175), depolymerization rates for alginic acid in various acids (251, 25U, 255), a pK of 2.95 for the dissociation constant of alginic acid in normal potassium chloride solution (208), absorption spectra of algin (202), and properties determined in connection with molecular weight studies, such as sedimentation and diffusion velocity and partial specific volumes (209),... 

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