title: Protein separation by nonsynchronous coil planet centrifuge with aqueous-aqueous polymer phase systems.
abstract: Counter-current chromatographic separation of proteins was performed using a rotary-seal-free nonsynchronous coil planet centrifuge (CPC) fabricated in our laboratory. This apparatus has a unique feature that allows a freely adjustable rotational rate of the coiled separation column at a given revolution speed. The separation was performed using a set of stable proteins including cytochrome c, myoglobin and lysozyme with two different types of aqueous-aqueous polymer phase systems, i.e., PEG (polyethylene glycol) 1000-dibasic potassium phosphate, and PEG 8000-dextran T500 in 5 mM potassium phosphate buffer. Using a set of multilayer coiled columns prepared from 0.8 mm I.D. PTFE tubing with different volumes (11, 24, 39 ml), the effect of the column capacity on the partition efficiency was investigated under a given set of experimental conditions. Among these experiments, the best separation of proteins was attained using the 39 ml capacity column with a 12.5% (w/w) PEG 1000-12.5% (w/w) dibasic potassium phosphate system at 10 rpm of coil rotation under 800 rpm. With lower phase mobile at 0.2 ml/min in the head-to-tail elution, the resolution between cytochrome c and myoglobin was 1.6 and that between myoglobin and lysozyme, 1.9. With upper phase mobile in the head-to-tail elution, the resolution between lysozyme and myoglobin peaks was 1.5. In these two separations, the stationary phase retention was 35.0 and 33.3%, respectively. Further studies were carried out using a pair of eccentric coil assemblies with 0.8 mm I.D. PTFE tubing at a total capacity of 20 ml. A comparable resolution was obtained using both lower and upper phases as a mobile phase in a head-to-tail elution. The results of our studies demonstrate that the nonsynchronous CPC is useful for protein separation with aqueous-aqueous polymer phase systems.
authors.affiliation: College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan. kshino@pha.nihon-u.ac.jp
authors.forename: Kazufusa
authors.initials: K
authors.lastname: Shinomiya
authors.forename: Yozo
authors.initials: Y
authors.lastname: Kabasawa
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Yanagidaira
authors.forename: Haruo
authors.initials: H
authors.lastname: Sasaki
authors.forename: Minoru
authors.initials: M
authors.lastname: Muto
authors.forename: Tadashi
authors.initials: T
authors.lastname: Okada
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Ito
mesh_terms: D002498:Centrifugation; D003377:Countercurrent Distribution / Q000379:methods*; D007202:Indicators and Reagents; D011108:Polymers / Q000737:chemistry*; D011506:Proteins / Q000302:isolation & purification*; D014867:Water
chemical_list: D007202:Indicators and Reagents; D011108:Polymers; D011506:Proteins; D014867:Water