Dye-ligand Aqueous Two-phase Systems

  • G. Johansson


It is possible to generate two-phase liquid-liquid systems in which both phases have high concentrations of water. One way is to dissolve two polymers (differing in their chemical structure) together in water (Albertsson et al., 1981; Walter et al., 1985; Albertsson, 1986). Between these two water-rich layers proteins can be partitioned. Differences in the partitioning make it possible to resolve protein mixtures by liquid-liquid extraction. Many pairs of water-soluble polymers give rise to two-phase systems but their usefulness is often limited by high viscosity and extreme separation time. The most widely used two-phase systems have, until now, been those composed of dextran and polyethylene glycol (PEG). This chapter is therefore limited to this kind of aqueous two-phase system. Triazine dyes, covalently bound to the polymers, have been included in the systems to obtain selective liquid-liquid extraction of dye-binding proteins from mixtures such as cell homogenates.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Albertsson, P.-Å. (1986). Partition of Cell Particles and Macromolecules, 3rd edn, Wiley, New YorkGoogle Scholar
  2. Albertsson, P.-Å., Andersson, B., Larsson, C. and Åkerlund, H.-E. (1981). Phase partition—a method for purification and analysis of cell organelles and membrane vesicles. Methods Biochem. Anal., 28, 115CrossRefGoogle Scholar
  3. Albertsson, P.-Å., Sasakawa, S. and Walter, H. (1970). Cross partition and isoelectric points of proteins. Nature, Lond., 228, 1329CrossRefGoogle Scholar
  4. Axelsson, C.-G. and Shanbhag, V. P. (1976). Histone-hydrocarbon interaction. Partition of histones in aqueous two-phase systems containing poly-(ethyleneglycol)-bound hydrocarbons. Eur. J. Biochem., 71, 419CrossRefGoogle Scholar
  5. Birkenmeier, G., Tschechonien, B. and Kopperschlager, G. (1984a). Affinity chromatography and affinity partition of human serum pre-albumin using immobilized Remazol Yellow GGL. Evidence that albumin increases binding of pre-albumin to the dye. FEBS Lett., 174, 162CrossRefGoogle Scholar
  6. Birkenmeier, G., Usbeck, E. and Kopperschläger, G. (1984b). Affinity partitioning of albumin and β-fetoprotein in an aqueous two-phase system using poly-(ethylene glycol)-bound triazine dyes. Analyt. Biochem., 136, 264CrossRefGoogle Scholar
  7. Cordes, A. (1985). Affinitätsverteilung—Theoretische Aspekte sowie die Auslegung eines darauf basierenden Verfahrens zur Reinigung der Formiat-Dehydrogenase von Candida boidinii. Thesis, BraunschweigGoogle Scholar
  8. Flanagan, S. D. and Barondes, S. H. (1975). Affinity partitioning—a method for purification of proteins using specific polymer-ligands in aqueous polymer two-phase systems. J. Biol. Chem., 250, 1484Google Scholar
  9. Gemeiner, P., Mislovičová, D., Zemek, J. and Kuniak, L. (1981). Anthraquinone-triazine derivatives of polysaccharides. Relation between structure and affinity to lactate dehydrogenase. Coll. Czech. Chem. Commun., 46, 419CrossRefGoogle Scholar
  10. Huse, K., Kopperschläger, G. and Hofmann, E. (1976). Differences in the degradation of yeast phosphofructokinase by proteinases A and B from yeast. Biochem. J., 155, 721CrossRefGoogle Scholar
  11. Johansson, G. (1970a). Partition of salts and their effects on partition of proteins in a dextran-poly(ethylene glycol)-water two-phase system. Biochim. Biophys. Acta, 221, 387CrossRefGoogle Scholar
  12. Johansson, G. (1970b). Studies on aqueous dextran-poly(ethylene glycol) two-phase systems containing charged poly(ethylene glycol). I. Partition of albumins. Biochim. Biophys. Acta, 222, 381CrossRefGoogle Scholar
  13. Johansson, G. (1974a). Effects of salts on the partition of proteins in aqueous polymeric two-phase systems. Acta Chem. Scand., B28, 873CrossRefGoogle Scholar
  14. Johansson, G. (1974b). Partition of proteins and micro-organisms in aqueous biphasic systems. Molec. Cell. Biochem., 4, 169CrossRefGoogle Scholar
  15. Johansson, G. (1976). The effect of poly(ethylene glycol) esters on the partition of proteins and fragmented membranes in aqueous biphasic systems. Biochim. Biophys. Acta, 451, 517CrossRefGoogle Scholar
  16. Johansson, G. (1978). Comparison of two aqueous biphasic systems used for partition of biological material. J. Chromatogr., 150, 63CrossRefGoogle Scholar
  17. Johansson, G. (1984). Affinity partitioning of enzymes. Methods Enzymol., 104, 356CrossRefGoogle Scholar
  18. Johansson, G. (1985). Determination of ionic charge by liquid-liquid partition. J. Chromatogr., 322, 425CrossRefGoogle Scholar
  19. Johansson, G. and Andersson, M. (1984a). Liquid-liquid extraction of glycolytic enzymes from bakers’ yeast using triazine dye ligands. J. Chromatogr., 291, 175CrossRefGoogle Scholar
  20. Johansson, G. and Andersson, M. (1984b). Parameters determining affinity partitioning of yeast enzymes using polymer-bound triazine dye ligands. J. Chromatogr., 301, 39CrossRefGoogle Scholar
  21. Johansson, G., Andersson, M. and Akerlund, H.-E. (1984). Counter-current distribution of yeast enzymes with polymer-bound triazine dye affinity ligands. J. Chromatogr., 298, 483CrossRefGoogle Scholar
  22. Johansson, G., Hartman, A. and Albertsson, P.-Å. (1973). Partition of proteins in two-phase systems containing charged poly(ethylene glycol). Eur. J. Biochem., 33, 379CrossRefGoogle Scholar
  23. Johansson, G. and Joelsson, M. (1985a). Preparation of Cibacron Blue F3G-A (polyethylene glycol) in large scale for use in affinity partitioning. Biotechnol. Bioengng, 27, 621CrossRefGoogle Scholar
  24. Johansson, G. and Joelsson, M. (1985b). Partial purification of glucose 6-phosphate dehydrogenase from bakers’ yeast by affinity partitioning using polymer-bound triazine dyes. Enzyme Microb. Technol., 7, 629CrossRefGoogle Scholar
  25. Johansson, G. and Joelsson, M. (1986). Liquid—liquid extraction of lactate dehydrogenase from muscle using polymer-bound triazine dyes. Applied Biochem. Biotechnol., 13, 15CrossRefGoogle Scholar
  26. Johansson, G., Joelsson, M. and Åkerlund, H.-E. (1985a). An affinity ligand gradient technique for purification of enzymes by counter-current distribution. J. Biotechnol., 2, 225CrossRefGoogle Scholar
  27. Johansson, G., Joelsson, M., Olde, B. and Shanbhag, V. P. (1985b). Affinity partitioning of biopolymers and membranes in Ficoll-dextran aqueous two-phase systems. J. Chromatogr., 331, 11CrossRefGoogle Scholar
  28. Johansson, G., Kopperschläger, G. and Albertsson, P.-Å. (1983). Affinity partitioning of phosphofructokinase from bakers’ yeast using polymer-bound Blue F3G-A. Eur. J. Biochem., 131, 589CrossRefGoogle Scholar
  29. Kopperschläger, G. and Johansson, G. (1982). Affinity partitioning with polymer-bound Cibacron Blue F3G-A for rapid large-scale purification of phosphofructokinase from bakers’ yeast. Analyt. Biochem., 124, 117CrossRefGoogle Scholar
  30. Kopperschläger, G. and Johansson, G. (1985). Studies on the ATP-sensitivity of yeast phosphofructokinase by means of affinity partitioning using polymer bound Cibacron Blue F3G-A. Biomed. Biochim. Acta, 44, 1047Google Scholar
  31. Kopperschläger, G. and Lorenz, G. (1985). Interaction of yeast glucose 6-phosphate dehydrogenase with diverse triazine dyes: A study by means of affinity partitioning. Biomed. Biochim. Acta, 44, 517Google Scholar
  32. Kopperschläger, G., Lorenz, G. and Usbeck, E. (1983). Affinity partitioning in an aqueous two-phase system to the investigation of triazine dye—enzyme interactions. J. Chromatogr., 259, 97CrossRefGoogle Scholar
  33. Kroner, H. K., Cordes, A., Schelper, A., Morr, M., Bückmann, A. F. and Kula, M.-R. (1982). Affinity partition studied with glucose-6-phosphate dehydrogenase in aqueous two-phase systems in response to triazine dyes. In Affinity Chromatography and Related Techniques (ed. T. C. J. Gribnau, J. Visser and R. J. F. Nivard), pp. 491–501, Elsevier, AmsterdamGoogle Scholar
  34. Kula, M.-R., Johansson, G. and Bückmann, A. F. (1979). Large-scale isolation of enzymes. Biochem. Soc. Trans., 7, 1CrossRefGoogle Scholar
  35. Kula, M.-R., Kroner, K. H. and Hustedt, H. (1982). Purification of enzymes by liquid—liquid extraction. In Advances in Biochemical Engineering (ed. A. Fiechter), Vol. 24, pp. 73–118, Springer-Verlag, BerlinGoogle Scholar
  36. Menge, U., Morr, M., Mayr, U. and Kula, M.-R. (1983). Purification of human fibroblast interferon by extraction in aqueous two-phase systems. J. Appl. Biochem., 5, 75Google Scholar
  37. Schiemann, J. and Kopperschläger, G. (1985). Binding of higher plant NADH-dependent nitrate reductase to different triazine dyes. Plant Sci. Lett., 36, 205CrossRefGoogle Scholar
  38. Shanbhag, V. P. and Axelsson, C.-G. (1975). Hydrophobic interactions determined by partition in aqueous two-phase systems. Partition of proteins in systems containing fatty-acid esters of poly(ethylene glycol). Eur. J. Biochem., 60, 17CrossRefGoogle Scholar
  39. Shanbhag, V. P. and Johansson, G. (1974). Specific extraction of human serum albumin by partition in aqueous biphasic systems containing poly(ethylene glycol) bound ligand. Biochem. Biophys. Res. Commun., 61, 1141CrossRefGoogle Scholar
  40. Walter, H., Brooks, D. E. and Fisher, D. (Eds.) (1985). Partitioning in Aqueous Two-Phase Systems, Academic Press, New YorkGoogle Scholar
  41. Walter, H., Sasakawa, S. and Albertsson, P.-Å. (1972). Cross partition of proteins. Effect of ionic composition and concentration. Biochemistry, 11, 3880CrossRefGoogle Scholar

Copyright information

© The contributors 1987

Authors and Affiliations

  • G. Johansson

There are no affiliations available

Personalised recommendations