Effects of salts and the surface hydrophobicity of proteins on partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers

The partitioning of five well-characterised model proteins, bovine serum albumin (BSA), lysozyme, beta-lactoglobulin A, myoglobin and cytochrome c, in aqueous two-phase systems has been studied. As top phase polymers PEG (polyethylene glycol, 100% EO) and the thermoseparating ethylene oxide (EO)-propylene oxide (PO) random copolymers, Ucon 50-HE-5100 (50% EO, 50% PO) and EO(30)PO(70) (30% EO, 70% PO), respectively, were used. The top phase polymers are increasing in hydrophobicity with increasing content of PO. Reppal PES 200 (hydroxypropyl starch) was used as the bottom phase polymer. Phase diagrams for Reppal PES 200-PEG and Reppal PES 200-EO(30)PO(70) two-phase systems were determined. The partitioning of four salts with different hydrophobicity, and also the effect of the salts on protein partitioning in these systems, was studied. It was found that the partitioning of the salts followed the Hofmeister series. The partitioning of proteins with low surface hydrophobicity, myoglobin and cytochrome c, was little affected by hydrophobic polymers and salts. However, the partitioning of a protein with higher surface hydrophobicity, lysozyme, was strongly affected when polymer hydrophobicity was increased and a hydrophobic counterion was used. A protein with a relatively hydrophobic surface can be partitioned to a phase containing a thermoseparating EO-PO copolymer by using a hydrophobic counterion. The partitioning of lysozyme and cytochrome c in the polymer-water system formed after temperature-induced phase separation was also examined. Both proteins partitioned exclusively to the water phase. A separation of the protein and polymer was obtained by temperature-induced phase separation on the isolated phase containing the EO-PO copolymer. The partitioning data also indicated that the hydroxypropyl starch polymer had a weak negative charge.