Zonal centrifugation

Macmillan and coworkers51 105 106 purified pectinesterase produced by Clostridium multifermentans by using practically all of the available methods and materials (calcium phosphate gel, DEAE-cellulose, DEAE-, QEAE-, CM-, and SE-Sephadex, Sephadexes G-75, G-100, G-150, and G-200, Sepharose 4B, and zonal centrifugation). However, they could not separate pectinesterase from exo-pectate lyase, and, hence, they postulated that either (a) a complex of the two enzymes having an apparent molecular weight of 400,000 exists, or (b) the two enzymes are identical in their molecular species. On the basis of the mode of action of this pectinesterase in comparison with that of those from tomatoes and from Fusarium ox-ysporum, the existence of a complex of pectinesterase and exopectate lyase in Clostridium multifermentans appears to be the more probable. 

Zonal techniques may be used for the separation of a wide range of particles and macromolecules, e.g. mitochondria, nuclei, ribosomes and proteins. The technique may be used for bulk preparative work using a zonal rotor which is filled with a solvent gradient while running at a slow speed. The sample is similarly introduced and the rotor speed is then increased to the desired value. After centrifugation is complete, the contents are drawn off while the rotor is running slowly by displacing them with a more dense solution. 

After dissociation of the 70 S ribosome into its two subunits followed by zonal centrifugation for the separation and isolation of the 30 S and 50 S subunits on a preparative scale, the ribosomal proteins were extracted by acetic acid and then separated by cellulose ion exchange chromatography and by gel filtration on Sephadex in the presence of 6 M urea. In this way all the 53 individual ribosomal proteins have been isolated (Wittmann, 1974). Proteins prepared in this manner have been used for physical studies (Brimacombe et al., 1978 Wittmann, 1982) as well as for immunological investigations (Stoffler et al., 1980 Lake,... 

Specialty rotors permit ordinary botde centrifuges to achieve some of the results previously considered possible only in ultracentrifuges. A modified zonal rotor, shown in Figure 9, permits collection of sediment using continuous addition of feed and discharge of centrate. 

Fig. 11. (a) Differential centrifugation (pelleting), where time 1 < time 2 < time 3 < time, (b) Rate zonal separation in a swinging-bucket rotor, where tube 1 represents the density gradient solution, tube 2 the sample plus the gradient, and tube 3 the separation of sample particles under a centrifugal force, where... 

Studies of interdiffusion have been extended to polymer systems. Structured flow formation was detected for these systems in zonal centrifugation experiments 66,67). If a polymer solution is layered on top of a more dense solution containing a low molecular weight solute, then interdiffusion of the salt into the polymer solution eventually gives rise to density inversion, and droplets of polymer solution will sediment. This effect has often been described as droplet sedimentation or the turnover effect 68). 

In another approach subcellular fractionation of human PMNs, stimulated in vitro with phorbol myristate acetate or opsonized zymsan, was performed by rate-zonal centrifugation in sucrose gradients By varying the centrifugal force the NADPH oxidase could be separated from the activities of enzymes in the lysosomal granules but was similar to the distribution of markers for the plasma membrane, aryl phosphatase, alkaline phosphodiesterase I, and acid aryl phosphatase. 

Pfleger, R. C., Anderson, N. G. and Snyder, F. 1968. Lipid class and fatty acid composition of rat liver plasma membranes isolated by zonal centrifugation. Biochemistry 7, 2826-2833. 

A comparison of differential and density gradient measurements. A Differential centrifugation in a medium of unchanging density. B Zonal centrifugation in a prepared density gradient. 

Carter, C.E., Wells, J.R. and Madnnis, A.J. (1972) DNA from anaerobic Ascaris lumbricoides and ffymenolepis diminuta mitochondria isolated by zonal centrifugation. Biochimica et Biophysica Acta 262, 135-144. 

Rate-zonal In rate-zonal centrifugation the sample is layered at the top of a centrifuge tube... 

Proteins with large differences in molecular mass can be separated by rate-zonal centrifugation using a gradient of a dense material such as sucrose. 

Boone et al. (1968) centrifuge cells through a discontinuous 10-20% Ficoll gradient made up in Eagle s minimum essential medium modified for suspension (i.e. lacking calcium and bicarbonate and containing 10 times the normal phosphate concentration). They use an A-1X zonal centrifuge rotor and spin for 1 h at 1000 r.p.m. at 20°C, and obtained clear separation of different cell types (HeLa and rabbit thymocytes). 

This involves the use of a special rotor and the adaptation of the centrifuge for constant flow as for the zonal rotor. This is expensive, but once installed, reproducible separations of 108-109 cells can be achieved in under an hour and cells of all sizes (rather than just the smallest) are obtained. It is particularly suited to the isolation of Gl-phase cells from suspension cultures for which mitotic detachment procedures are not applicable. The separation chamber is kite-shaped with the buffer solution entering at the acute point on the rim of the centrifuge and leaving at the obtuse point towards the centre of rotation (Fig. 11.2). Thus the centrifugal forces on particles within the chamber are countered by the centripetal flow of buffer and particles come to equilibrium within the chamber. When equilibrium has been reached, the samples are pumped out, by increasing the rate of buffer flow, and collected. Meistrich et al. (1977) obtained 3 fractions of L-P59 mouse fibroblasts which were over 90% Gl-phase, 70% S-phase cells and 60% G2 + M-phase cells, respectively. 

While there are many practical problems in realizing the theoretical power of sedimentation, major advances have accompanied the development of the disc centrifuge and of the zonal rotor [43-45]. 

N. G. Anderson, Ed., Zonal Centrifugation, National Cancer Institute Monograph No. 21, 1966. 

Figure 6.8 Diagrammatic illustration of zonal and isopycnic centrifugation.

For zonal density gradient centrifugation there are a number of media that can be used, however the most common of these is sucrose whereas the most common medium for isopycnic density gradient centrifugation is caesium chloride (CSCI2). 

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