Biochemical Applications

CCD has been used for fractionation of a number of biochemical substances and cellular particles as well as cells and viruses. Peptides, proteins and nucleic acids have been fractionated by using aqueous two-phase systems composed of water and the two polymers dextran and poly(ethylene glycol) (PEG). The partition coefficients of the solutes can be adjusted by addition of various salts to the two-phase system and by adjusting the pH value. More selective adjustment of the partitioning has been carried out by linking an affinity ligand to one of the phase-forming polymers which then is concentrated in one of the phases.


Procion yellow HE-3G PEG

/ i i I.^'n.

Figure 7 Countercurrent distribution of proteins and cell organelles. (A) Distribution of the enzymes hexokinase (—), 3-phosphoglyceratekinase (—), and phosphofructokinase

( ) when an extract of baker's yeast was applied to a CCD

with 55 transfers. The textile dyes Procion olive MX-3G or Procion yellow HE-3G were used as PEG-bound affinity ligands enriched in the mobile upper phase. Composition of two-phase system: 88% (w/w) water, 7% (w/w) dextran 500, 5% PEG with M = 8000, including dye-PEG, 1% of total PEG 50mmolL~1 sodium phosphate buffer, pH 7.0, 0.2 mmol L~1 ethylenediamine-tetraacetic acid and 5 mmol L~1 2-mercaptoethanol. Volume ratio, 1.5. Temperature, 3°C. A centrifugal CCD apparatus was used with 5 min shaking and 3 min settling (centrifugation). (Adapted from Johansson G, Joelsson M and Akerlund H-E (1984) Journal of Chromatography 298: 483, by permission.) (B) Fractionation of photosynthetic particles, chloroplasts, from spinach using a thin-layer CCD apparatus with 56 transfers and a dex-tran-PEG two-phase system. Peak I, intact chloroplasts surrounded by their envelope; peak II, naked thylakoid membranes (class II chloroplasts); peak III, choloroplasts surrounded by a 'bag' of plasma membrane also containing cytoplasm, mitochondria and peroxisomes. (From Larsson C, Collin C and Albertsson P-A (1971) Biochimica BiophysicaActa 245: 425, by permission.)

Biological membranes, cell organelles, whole cells and viruses can be fractionated by CCD in the same kind of systems. In this case, however, the particles partition between the two liquid phases and the interface between them. The CCD is therefore usually carried out using a stationary interface. This is achieved by using a smaller volume of the lower phase than is needed to fill the lower cavities. Therefore, a portion of the upper phases will also be stationary. The G value satisfying eqn [5] is in this case defined as the amount of a pure compound, at equilibrium, in the mobile part of the upper phase divided by the amount of the compound in the rest of the system (stationary upper phase, interface and lower phase). Examples of CCD of proteins and of chloro-plasts, the photosynthetic organelle in green plant cells, are given in Figure 7.

See also: II/Chromatography: Countercurrent Chromatography and High-Speed Countercurrent Chromatography: Instrumentation.

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