Band ultracentrifugation

Band ultracentrifugation is also a sedimentation velocity method, but here the particles sediment in a mixed solvent system (e.g., a salt solution). First a density gradient is produced by ultracentrifugation of the solvent system alone. Then the solute solution is added at the cell meniscus. The individual components sediment in bands which are no longer contaminated by the other components. The relatively weak density gradient is solely... 

Figure 9-15. Kinds of preparative ultracentrifugation methods. (S) Normal ultracentrifugation, (B) band ultracentrifugation in a stabilized gradient, (I) isopycnic zone centrifugation p is the density of the gradient-forming substance (O) high molar mass, low density ( ) low molar mass, high density.

Purified preparations of alkaline phosphatase from E. coli, judged homogeneous when examined in the analytical ultracentrifuge, contain several isozymes, because several bands which contain enzymic activity are obtained in starch-gel and disc-gel electrophoresis. Although most workers find three bands (38, 39, 41, 43, 69), four (44) and five (70) equally spaced bands have been found. 

TABLE 3. Chlorophyll-protein composition of the green bands separated by sucrose gradient ultracentrifugation of photosystem II. band... 

If a further purification is desired, then the supernatant (above) can be subjected to ammonium sulfate fractionation, gel filtration on Sephadex G-200, and finally on a-aminopropane-agarose affinity column. On polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, the final product migrated as a single band with an estimated molecular weight of 113,000. Upon sedimentation equilibrium velocity ultracentrifugation, an estimated molecular weight value of near 117,000 was obtained. 

Cytochrome 62 is found as a soluble protein in the autolysates of Sac-charomyces cerevisiae. The crystalline preparations of Appleby and Morton 278) were shown to sediment as a single peak in the ultracentrifuge. Minimum molecular weight based on amino acid analysis and a heme extinction coefficient of 232 mM cm was calculated to be 53,000 283). The heme extinction coefficient was then corrected to 183 mM- cm-, and the minimum molecular weight per mole of heme recalculated to be 58,600 284). It was concluded that cytochrome 6 is a tetrameric structure. This conclusion agreed with the results of X-ray diffraction studies on type I and type II crystals, which indicated molecular weights of 235,000 10,000 and 234,000 8,000, respectively, for these two preparations of cytochrome 2 285). The oxidized and reduced spectral bands of cytochrome ba are given in Table XIV. 

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