Centrifugation molecular weight determinations

Ultracentrifugation is widely used for the purification, separation, and molecular-weight determination of proteins. A centrifugal field, up to 500,000 times that of gravity, is applied to the solution, and molecules move downward in the field according to their mass and size. 

Three histone-specific acetyltransferases have been partially purified and characterized from rat thymus nuclei (225). The enzymes were extracted from rat thymus nuclei by sonication in the presence of 1M ammonium sulfate and separated into two active fractions (A and B) by DEAE-cellulose chromatography. Fraction B was further separated into two active fractions (Bi and B2) by gel filtration on Sephadex G-200. Each fraction was then purified further by chromatography on hydroxyapatite. The molecular weights, determined by Sephadex G-200 and by sucrose density gradient centrifugation, were 99,000, 110,000, and 92,000 for enzymes A, Bi, and B2, respectively. All three enzymes required acetyl CoA as acetate donor, and the activity of the enzymes was inhibited by p-chloromercuribenzoate. Acetyltransferase A preferentially acetylated histone I (FI) and also poly-L-lysine. Acetyltransferase Bi and B2 preferred histone H4 (other names IV, F2al) and did not acet-ylate poly-L-lysine and histone H3 (III, F3). In addition to c-N-acetyl-lysine, two other unidentified amino acid derivatives were obtained from a digest of histone H4 acetylated by the two B enzymes. 

In the case of polyelectrolytes another parameter has to be considered the degree of dissociation. Molecular weight determination in pure water can not be performed. The reason for this is that the viscosity is also very high in the dilute state and therefore the purification procedure (filtration, centrifugation) is difficult to perform. In addition, the difference between the scattering intensity of the light in the solvent and in the polymer solution is very small at this low concentration, which makes such measurements impossible. Therefore, all measurements have to be performed in a low... 

Solvent extraction is a commonly used laboratory technique for PHA recovery, especially for molecular weight determination. Usually, methanol- (or ethanol-) pretreated, lyophilized biomass is heated in chloroform imder reflux conditions for about 1 h. The polymer solution is recovered by filtration or centrifugation, and the polymer is precipitated in cold ethanol or methanol. The polymer may be further purified by redissolving in chloroform and reprecipitating in cold ethanol. It should be noted that about 25% of the original molecular weight (determined by chloroform extraction under ambient conditions) is lost when refluxed in chloroform for 1 h (68). 

For the determination of the sulfhydryl groups, the method of Habeed was used. Disulfide bridges were determinated by the same method with urea and DTT. The protein content was estimated by the method of Bradford with BSA as a standard. The molecular weight determinations were effected by molecular sieving over Sephadex G-200 and by sedimentation equilibrium. Analytical centrifugations experiments were performed with a Beckman model E ultracentrifuge equipped with an interferometric system for Mr determination. For the active enzyme sedimentation constant determination, we have used the method of Cohen and Mire (CEA method). 

With the a-CD-PEG complexes isolated by filtration or centrifugation, the yields of complexes were determined as a function of PEG molecular weight, assuming that two ethylene glycol units are bound to one a-CD, i.e., the stoichiometric ratio is 2 1, and the results shown in Fig. 3 were obtained. It is... 

Selected entries from Methods in Enzymology [vol, page(s)] Association constant determination, 259, 444-445 buoyant mass determination, 259, 432-433, 438, 441, 443, 444 cell handling, 259, 436-437 centerpiece selection, 259, 433-434, 436 centrifuge operation, 259, 437-438 concentration distribution, 259, 431 equilibration time, estimation, 259, 438-439 molecular weight calculation, 259, 431-432, 444 nonlinear least-squares analysis of primary data, 259, 449-451 oligomerization state of proteins [determination, 259, 439-441, 443 heterogeneous association, 259, 447-448 reversibility of association, 259, 445-447] optical systems, 259, 434-435 protein denaturants, 259, 439-440 retroviral protease, analysis, 241, 123-124 sample preparation, 259, 435-436 second virial coefficient [determination, 259, 443, 448-449 nonideality contribution, 259, 448-449] sensitivity, 259, 427 stoichiometry of reaction, determination, 259, 444-445 terms and symbols, 259, 429-431 thermodynamic parameter determination, 259, 427, 443-444, 449-451. 

The data plotted in Figure 3.6 were obtained by the fractionation of a polydisperse polymer sample. Polydisperse polymers can be fractionated by a number of techniques. The most widely used technique is chromatography. Other methods include addition of a nonsolvent to a polymer solution, cooling a polymer solution, solvent evaporation, extraction, diffusion, and centrifugation. The molecular weight of the fractions may be determined using any of the classic techniques given in Table 3.3. 

An alternative description of protein-containing RMs was given by Levashov et al. [148] from ultra-centrifugation studies. Water-soluble dyes (picric acid) were used to determine the molecular weights and sedimentation behavior of the non-protein-containing and protein-containing RMs. The proposed model involved two regimes of Wg (i) at low Wg (where the inner reverse micellar diameter was less than the protein diameter), the protein created a new RM around itself such that the volume of the inner cavity of the filled RM was essentially the volume of the solubihzed protein and (ii) at water contents where... 

Some data for the molecular weight of cellulose of various origins determined by ultra-centrifuging cuprammonium solution, are given in Table 35. 

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