Affinity partition

Johansson, G. (1992) Affinity partitioning in PEG-containing two-phase systems. In Poly(Ethylene Glycol) Chemistry Biotechnical and Biomedical Applications (J.M. Harris, ed.), pp. 73-84. Plenum, New York. 

Other applications besides purification of biomolecules are affinity partitioning (Labrou, 1994), extractive bioconversions (Andersson, 1990 Kaul, 1991 Zijlstra, 1998), liquid-liquid partition chromatography, and analytical assays. Although industrial applications of aqueous two-phase systems have not gained widespread use to date, practical and economic feasibility has been proved (Tjemeld, 1990 Cunha, 2002). 

Johansson G (1998), Affinity partitioning of proteins using aqueous two-phase systems, In Janson JC, Ryden L (Eds), Protein Purification Principles, High-Resolution Methods, and Applications, 2nd Ed., Wiley-VCH, Weinheim, pp. 443-460. 

TABLE 11 Affinity Partitioning of Biomolecules in Aqueous Two-Phase Systems... 

Harris, J. M., and Yalpani, M. (1985). Polymer-ligands used in affinity partitioning and their synthesis. In Partitioning in Aqueous Two-Phase Systems Theory, Methods, Uses, and Applications to Biotechnology (H. Walter, D. E. Brooks, and D. Fisher, eds.), pp. 589-626. Academic Press, New York. 

These measurements were conducted in ATP system consisting of 5.1% (w/w) PEG 8000 and 10% (w/w) dextran 70 in phosphate buffer at 4°C. Under these conditions, the volume ratio of PEG (light) to dextran (heavy) phases was 3 7. The composition (w/w) of PEG phase was 9.25% PEG and 0.52% dextran. The composition of the dextran phase was 0.54% PEG and 21.1% dextran. The phase composition was unaffected by NaCl concentration in the range of 0-5 M. The partition coefficient is defined as ratio of concentration in PEG phase to that in dextran phase. The triazine dye, Cibacron blue FGF, was ligated to PEG for affinity partitioning. 

Jaeske, A., Fuertste, J. P., Erdmann, V. A., and Cech, D. (1994). Hybridization-based affinity partitioning of nucleic acids using PEG-coupled oligonucleotides. Nucleic Acids Res. 22, 1880 1884. 

Affinity Partitioning. A new area that has only just begun to be... 

The hydrophobic effect refers to the favorable interactions between nonpolar surfaces immersed in water. These interactions are considered to provide the driving force for protein folding (44) and to make a major contribution to the stability of protein tertiary stractures. The hydrophobic effect also plays an important role in protein interactions (45). The hydrophobicity of protein surfaces has been studied experimentally by affinity partitioning of proteins (46). Theoretical studies have shown that the presence of hydrophobic patches on the surfaces of proteins correlates with protein binding sites (47 9). 

The equations used in our study of the thermodynamics and kinetics of thermal electron reactions using the ECD and NIMS are presented. The ECD and NIMS methods were developed in our laboratories. These are used to determine the rate constants, electron affinities, partition function ratios, and bond dissociation energies of molecules and energies for the formation of complexes of anions. The general kinetic model for the ECD and negative-ion mass spectrometry is presented. Molecules will be classified using example data. 

Mattiasson, B. Ling, T.G.I. Efforts to integrate affinity interactions with conventional separation technologies affinity partition using biospecific chromatographic particles in aqueous two-phase systems. J. Chromatogr. 1986, 376, 235-243. 

In an isolation step, where yield and concentration are more important than purity, the adsorption mechanism can be considered an on/off process, and several alternative contacting schemes can be used. Ligands have been bound to magnetized particles (137, 138) for continuous countercurrent adsorption in magnetically stabilized fluidized beds. Ligands attached to liquid perfluorocarbons (143), to dextran and related polymers (144), or incorporated into liposomes (145), or reversed micelles (146) may be used for biospecific liquid-liquid extraction or "affinity partitioning". Ligands have also been attached to surfactants and biopolymers for selective precipitation of dilute protein species (147, 148). 

Kopperschlager, G. Lorenz, G. Usbeck, E. "Application of Affinity Partitioning in an Aqueous Two-Phase System to the Investigation of Triazine Dye-Enzyme Interactions" /. Chromatography. 1983, 259, pp 97-105. 

Firary, M. Carlson, A. "Affinity partitioning of Acid Proteases in the Hydrox)q)ropyldextran-dextran Aqueous Two-Phase System" presented at the AIChE Summer Meeting, Boston, M A, August 1986. 

The main drawback to the widespread use of polymer-polymer aqueous two-phase extraction has been the high cost of fractionated dextran. Crude dextran has been used with some success for the purification of enzymes but is much too viscous for many applications. Conversely, polymer-salt systems have relatively low viscosities, separate rapidly, and are inexpensive. Unfortunately, they lack selectivity and cannot be used for affinity partitioning in most cases since the high salt concentrations interfere with the protein-ligand interaction. The starch derivatives are reasonable alternatives for bottom phase polymers but have been hampered by low solubilities and the tendency for gel formation. Tjemeld has reported that chemically modified starches i.e. hydroxypropyl starch... 

The MD/PEG system offers the combined advantages of low-cost, reduced lower phase viscosities and high density differences for inexpensive polymer-polymer affinity partitioning. When coupled with low-cost affinity ligands i.e. triazine dyes, two-phase aqueous affinity partitioning could be used as the first step in a separation train for the recovery of industrially important enzymes. The bottom phase, which is generally considered to be a waste stream and non-recyclable if dextran or salt is used could be used as a substrate for additional fermentations if maltodextrin is used, thereby aiding the overall economics of the process. 

Affinity partitioning. In some cases, it may not be possible to separate one protein out of a complex mixture by means of spontaneous partitioning in an aqueous two-phase system. Then affinity interactions may be utilized. When first described, affinity partitioning was used for purification of membrane vesicles (26), and has since been exploited in a broad spectrum of applications. 

Table III. Examples of affinity partitioning in aqueous two-phase systems.

By this technique of combining affinity partitioning with affinity chromatographic elution, the advantages of the two procedures were combined. The rapid and effective removal of cell debris in the extraction procedure, and finally, the efficient elution procedure of the affinity chromatographic step was achieved. 

Extraction in aqueous two-phase systems is said to be an operation that is easy to scale up (33). This is also true for affinity partitioning and is clearly illustrated by the process for purification of formate dehydrogenase (34, 35). In this study, small scale experiments gave an overall enzyme yield of 74%. When scaled up by a factor of 40 000, the yield was 70%, thus demonstrating the feasibility of evaluating the performance of extraction process on a small scale. 

A reason why the PEG/salt systems have not been used more extensively is that the high ionic strength severely influences the affinity interactions when affinity partitioning is to be exploited. However, in applications where spontaneous partitioning is sufficient, these systems are often used. Recently, the use of starch derivatives named as Reppal PES has been reported (32) that are useful in forming aqueous two-phase systems. The properties of these new polymers resemble those of dextran. 

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