EFFECTS OF SALTS AND THE SURFACE HYDROPHOBICITY OF PROTEINS ON PARTITIONING IN AQUEOUS 2-PHASE SYSTEMS CONTAINING THERMOSEPARATING ETHYLENEOXIDE-PROPYLENE OXIDE COPOLYMERS

The partitioning of five well-characterised model proteins, bovine ser um albumin (BSA), lysozyme, beta-lactoglobulin A, myoglobin and cytoch rome c, in aqueous two-phase systems has been studied. As top phase po lymers PEG (polyethylene glycol, 100% EO) and the thermoseparating eth ylene oxide (EO)-propylene oxide (PO) random copolymers, Ucon 50-HE-51 00 (50% EO, 50% PO) and EO(30)PO(70) (30% EO, 70% PO), respectively, w ere used. The top phase polymers are increasing in hydrophobicity with increasing content of PO. Reppal PES 200 (hydroxypropyl starch) was u sed 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. Th e partitioning of four salts with different hydrophobicity, and also t he effect of the salts on protein partitioning in these systems, was s tudied. It was found that the partitioning of the salts followed the H ofmeister series. The partitioning of proteins with low surface hydrop hobicity, myoglobin and cytochrome c, was little affected by hydrophob ic polymers and salts. However, the partitioning of a protein with hig her surface hydrophobicity, lysozyme, was strongly affected when polym er 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 hydr ophobic counterion. The partitioning of lysozyme and cytochrome c in t he polymer-water system formed after temperature-induced phase separat ion was also examined. Both proteins partitioned exclusively to the wa ter phase. A separation of the protein and polymer was obtained by tem perature-induced phase separation on the isolated phase containing the EO-PO copolymer. The partitioning data also indicated that the hydrox ypropyl starch polymer had a weak negative charge.