title: Affinity partitioning. A method for purification of proteins using specific polymer-ligands in aqueous polymer two-phase systems.
abstract: We describe a method, called affinity partitioning, for the purification of proteins containing specific ligand binding receptor sites. This method adds specificity to the procedures for protein purification with aqueous polymer two-phase systems by introduction of a polymer derivative, coupled to an appropriate ligand. The addition of a polymer-ligand that partitions predominantly into one phase shifts the protein that binds this substance to the same phase. By performing countercurrent distribution in the presence of a polymer-ligand, the protein that binds the polymer-ligand can be separated from a heterogenous mixture. One example of affinity paritioning used dextran as the polymer-ligand. Dextran was chosen since it is a constituent of the most commonly used system for partitioning proteins. In a dextran-poly(ethylene oxide) system, concanavalin A bound dextran and partitioned predominantly into the dextran-rich phase. The addition of the specific competitor, D-mannose, displaced the partition coefficient toward unity, while the application of L-fucose, a noncompetitor, had little effect. Application of affinity partitioning to the purification of another protein required the synthesis of a specific polymer-ligand. To study this we synthesized dinitrophenyl-poly-(ethylene oxide), which binds specifically to S-23 myeloma protein. Addition of dinitrophenyl-poly(ethylene oxide) to the dextran-poly(ethylene oxide) phase system shifted the S-23 myeloma protein into the poly(ethylene oxide)-rich phase. epsilon-N-dinitrophenyl-L-lysine, by competing with binding of dinitrophenyl-poly(ethylene oxide), antagonized the latter's effect on the partition coefficient of S-23 myeloma protein. By adding various amounts of dinitrophenyl-poly-(ethylene oxide), we correlated the partition coefficient with concentration of polymer-ligand. A model of the action of polymer-ligand derivatives on the partition coefficient, derived from thermodynamic considerations, was found to be consistent with the experimental data relating the concentration of polymer-ligand and partition coefficient. Affinity partitioning should prove to be a useful complement to affinity chromatography in the purification of mixtures of proteins. Since cells and subcellular particles may be purified with aqueous polymer two-phase systems, affinity partitioning might be applied to their fractionation by using polymer-ligands specific for unique surface receptors.
mesh_terms: D000818:Animals; D002850:Chromatography, Gel; D003208:Concanavalin A / Q000302:isolation & purification; D003377:Countercurrent Distribution / Q000379:methods*; D003911:Dextrans; D004140:Dinitrophenols; D005029:Ethylenediamines; D008024:Ligands; D008433:Mathematics; D009194:Myeloma Proteins / Q000302:isolation & purification; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification*; D011817:Rabbits; D012685:Sepharose; D012712:Serum Globulins / Q000302:isolation & purification
chemical_list: D003911:Dextrans; D004140:Dinitrophenols; D005029:Ethylenediamines; D008024:Ligands; D009194:Myeloma Proteins; D011506:Proteins; D012712:Serum Globulins; D003208:Concanavalin A; D012685:Sepharose