Partitioning of biomolecules in thermoseparating polymer-water two-phase systems

Abstract: The partitioning behaviour of different types of molecules in thermoseparating two-phase systems has been studied. A thermoseparating two phase system was formed by heating a water solution, which contained a thermoseparating polymer, above the cloud point temperature. The thermoseparating polymers studied were: random copolymers of ethylene oxide and propylene oxide (EOPO-polymers), and ethyl(hydroxy ethyl)cellulose. The phase behaviour of ternary systems composed of a thermoseparating polymer, water and a cosolute was investigated. Strongly hydrophobic and hydrophilic cosolutes caused a decrease of cloud point temperature of the system compared with corresponding cosolute-free system. Separation of amino acids and peptides in the thermoseparating polymer/water two phase system was obtained. The more hydrophobic, aromatic amino acids or peptides were partitioned to the polymer rich phase, while most other amino acids, lysinyl- and glycinyl peptides partitioned to the water rich phase. The influence of salts on the partitioning of charged amino acids and peptides was strong. The positively charged polypeptide poly(Lys,Trp) was quantitatively partitioned to the polymer rich- or the water rich phase upon the addition of NaClO4 and Na2SO4, respectively. The partitioning of albumin and lysozyme in an aqueous two phase system was investigated. The top and bottom phases in this two phase system contained a thermoseparating EOPO-polymer and dextran, respectively. Albumin and lysozyme were partitioned to the top phase by the addition of the salts triethylammonium phosphate and NaClO4, respectively. By heating the top phase separately, a new two phase system was obtained composed of a water rich top phase and a polymer rich bottom phase. In this system the proteins were quantitatively partitioned to the water rich phase. Model calculations based on the Flory-Huggins theory of polymer solutions reproduced qualitatively the experimental results