Extraction and separation of a lysine-rich protein by formation of supramolecule between crown ether and protein in aqueous two-phase system

Abstract

The macrocyclic calixarenes and crown ethers have recently been found to form hydrophobic complexes with the cationic protein cytochrome c (Cyt- c), by recognizing lysine residues on the protein surface. In the present study, it was found that the distribution of cytochrome c in Li 2SO 4/PEG aqueous two-phase system (ATPS) can be controlled by complexation with the crown ether dicyclohexano-18-crown-6 (DCH18C6). The protein was quantitatively extracted into the PEG-rich phase in the presence of DCH18C6 and perchlorate ion. Of various crown ethers and their analogues that were investigated, only DCH18C6 was able to extract cytochrome c into the PEG-rich phase. Extraction of cytochrome c in the ATPS using DCH18C6 is complete within 5 min. Cytochrome c complexed with DCH18C6 in the PEG-rich phase was quantitatively recovered into a salt-rich phase using K 2SO 4 by ion exchange of potassium ion and cationic protein in the cationic protein complex with DCH18C6. Selective extraction of cationic proteins was demonstrated in the ATPS. Under optimum conditions, the lysine-rich protein cytochrome c was selectively extracted over other cationic proteins using DCH18C6.