Aqueous micellar two-phase system composed of hyamine-type hydrophobically modified ethylene oxide and application for cytochrome P450 BM-3 separation

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2007-06, Vol.852 (1), p.167-173
• Main Authors: Deng, Gang, Lin, Dong-Qiang, Yao, Shan-Jing, Mei, Le-He
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Aqueous micellar two-phase system; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Benzethonium - analogs & derivatives; Benzethonium - chemistry; Biological and medical sciences; Cytochrome P-450 Enzyme System - isolation & purification; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450 BM-3; Electrostatic interaction; Ethylene Oxide - chemistry; Fundamental and applied biological sciences. Psychology; General pharmacology; Hydrogen-Ion Concentration; Hydrophobically modified ethylene oxide; Medical sciences; Micellar aggregate; Micelles; Mixed Function Oxygenases - isolation & purification; Mixed Function Oxygenases - metabolism; NADPH-Ferrihemoprotein Reductase; Pharmacology. Drug treatments
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2007.01.008

The hydrophobically modified ethylene oxide polymer, HM-EO, was modified with an alkyl halide to prepare a hyamine-type HM-EO, named N-Me-HM-EO, which could be used for forming N-Me-HM-EO/buffer aqueous micellar two-phase system. The critical micelle concentration of N-Me-HM-EO solution and the phase diagrams of N-Me-HM-EO/buffer systems were determined. By using this novel aqueous micellar two-phase system, the separation of cytochrome P450 BM-3 from cell extract was explored. The partitioning behavior of P450 BM-3 in N-Me-HM-EO/buffer systems was measured. The influences of some factors such as total proteins concentration, pH, temperature and salt concentration, on the partitioning coefficients of P450 BM-3 were investigated. Since the micellar aggregates in the N-Me-HM-EO enriched phase were positively charged, it was possible to conduct the proteins with different charges to top or bottom phases by adjusting pH and salt concentration in the system. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: the first extraction with N-Me-HM-EO/buffer system at pH 8.0, and the second extraction in the same system at pH 6.0. The recovery of P450 BM-3 was 73.3% with the purification factor of 2.5. The results indicated that the aqueous micellar two-phase system composed of hyamine modified polysoap has a promising application for selective separation of biomolecules depending on the enhanced electrostatic interactions between micelles and proteins.