cytochrome-c-t and potassium-phosphate

The partition efficiency of the double-spaced coil for eccentric and toroidal coils on countercurrent chromatographic separation of proteins was evaluated using the small-scale cross-axis coil planet centrifuge (CPC) equipped with circular and elliptic cylindrical columns. Standard cytochrome c, myoglobin and lysozyme samples were used for separation with the 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate system. In the circular column, the double-spaced eccentric coil yielded better peak resolution than the double-spaced toroidal coil, and the double-spaced eccentric coil yielded better peak resolution than the single-spaced eccentric coil. In the elliptic column, the double-spaced eccentric coil also produced better peak resolution than the double-spaced toroidal coil, but the single-spaced eccentric coil yielded better peak resolution than the double-spaced eccentric coil. The overall results indicated that the double-spaced eccentric coil for the circular column and the single-spaced eccentric coil for the elliptic column yielded better protein separation using the small-scale cross-axis CPC with aqueous two-phase solvent systems.

Topics: Centrifugation; Countercurrent Distribution; Cytochromes c; Muramidase; Myoglobin; Phosphates; Planets; Polyethylene Glycols; Potassium Compounds

Factors contributing to the different distribution behaviour of cytochrome c were investigated in a biphasic tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate and potassium phosphate buffer system, which shows a lower critical solution temperature. To change charge balance and hydrophobicity of cytochrome c, surface modification with a few modifier molecules was applied. Surface charge and hydrophobicity affected the distribution behavior of chemically modified cytochrome c in the tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate and potassium phosphate buffer biphasic system. The distribution ratio into tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate decreased with decreasing isoelectric point of cytochrome c. Furthermore, cytochrome c possessing a low isoelectric point showed different distribution ratio depending on surface hydrophobicity. Taken together, these findings indicate that isoelectric point and surface hydrophobicity of cytochrome c are important factors controlling the distribution behavior in temperature sensitive biphasic systems.

Topics: Animals; Cytochromes c; Heart; Horses; Hydrophobic and Hydrophilic Interactions; Ionic Liquids; Phosphates; Potassium Compounds; Solutions; Surface Properties; Temperature

Protein separation was performed using the high-speed countercurrent chromatograph (HSCCC) at both synchronous and nonsynchronous type-J planetary motions. The partition efficiency was evaluated with two different column configurations, eccentric coil and toroidal coil, on the separation of a set of stable protein samples including cytochrome C, myoglobin and lysozyme with a polymer phase system composed of 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate. Better peak resolution was obtained by the eccentric coil than by the toroidal coil using either lower or upper phase as the mobile phase. The peak resolution was further improved using the eccentric coil by the nonsynchronous type-J planetary motion with the combination of 1066 rpm of column rotation and 1000 rpm of revolution.

Topics: Animals; Centrifugation; Countercurrent Distribution; Cytochromes c; Equipment Design; Humans; Motion; Muramidase; Myoglobin; Phosphates; Polyethylene Glycols; Polymers; Potassium Compounds; Proteins

A new spiral column assembly for analytical separation by counter-current chromatography is described. The column is made from a plastic spiral tube support which has 12 interwoven spiral grooves. The PTFE tubing of 1.6mm ID was first flattened by extruding through a narrow slit and inserted into the grooves to make 5 spiral layers with about 60ml capacity. The performance of the spiral column assembly was tested with separation of three stable protein samples including cytochrome C, myoglobin and lysozyme in a polymer phase system composed of polyethylene glycol 1000 and dibasic potassium phosphate each at 12.5% (w/w) in water. At 2ml/min, three protein samples were well resolved in 1h. The separation time may be further shortened by application of higher revolution speed and flow rate by improving the strength of the spiral tube support in the future.

Topics: Countercurrent Distribution; Cytochromes c; Muramidase; Myoglobin; Phosphates; Polyethylene Glycols; Potassium Compounds

A new spiral tube assembly was designed to improve the column capacity and partition efficiency for protein separation. This spiral tube assembly has greater column capacity than the original tubing because of an increase in radial grooves from 4 to 12 to accommodate more spiral layers and 12 narrow spots instead of 4 in each circular loop to interrupt the laminar flow that causes sample band broadening. Standard PTFE tubing (1.6mm ID) and the modified flat-twisted tubing were used as the separation column. The performances of both assemblies were compared for separating three stable test proteins including cytochrome c, myoglobin, and lysozyme using a two phase aqueous-aqueous solvent system composed of polyethylene glycol 1000 (12.5% w/w) and dibasic potassium phosphate (12.5% w/w). All samples were run at 1, 2, 3, and 5mL/min at both 800rpm and 1000rpm. The separation of these three protein samples produced high stationary phase retentions at 1, 2, and 3mL/min, yet separated efficiently at 5mL/min in 40min. After comparing the separation efficiency in terms of the peak resolutions, theoretical plate numbers, and separation times, it was determined that the flat-twisted tubing was more effective in separating these protein samples. In order to validate the efficacy of this novel assembly, a mixture of five protein samples (cytochrome c, myoglobin, ovalbumin, lysozyme, and hemoglobin) were separated, under the optimal conditions established with these three protein samples, at 1mL/min with a revolution speed of 1000rpm. There were high stationary phase retentions of around 60%, with effective separations, demonstrating the efficiency of the flat-twisted spiral tube assembly. The separation time of 6h was a limitation but can potentially be shortened by improving the strength of the column that will permit an increase in revolution speed and flow rate. This novel spiral separation column will allow rapid and efficient separation of mixtures with high yield of the constituent components.

Topics: Countercurrent Distribution; Cytochromes c; Hemoglobins; Indicators and Reagents; Muramidase; Myoglobin; Ovalbumin; Phosphates; Polyethylene Glycols; Potassium Compounds; Proteins; Solvents; Water

Compared with the conventional ionic liquids, amino acid ionic liquids are more biodegradable and biocompatible, and can enhance stability of biomaterials. In this work, amino acid ionic liquids 1-butyl-3-methylimidazolium L-serine ([C(4)mim][Ser]), 1-butyl-3-methylimidazolium glycine ([C(4)mim][Gly]), 1-butyl-3-methylimidazolium L-alanine ([C(4)mim][Ala]) and 1-butyl-3-methylimidazolium L-leucine ([C(4)mim][Leu]) have been synthesized. These ionic liquids are found to form aqueous two-phase systems (ATPSs) by the salted-out of K(3)PO(4) in aqueous solutions. Phase diagram of the ATPSs and the Gibbs energies of transfer of methylene group from the bottom salt-rich phase to the top ionic liquid-rich phase have been determined at 298.15K and pH 14, and the effect of anionic structure of the ionic liquids on phase formation of the ATPSs and the relative hydrophobicity between the top and the bottom phases are then examined. In order to understand the effect of relative hydrophobicity of the phases in equilibrium in the ATPSs on the extraction/separation capability of biomolecules, the partition coefficients of cytochrome-c (as a model biomolecule) in the ATPSs are measured by spectrophotometry. It is suggested that hydrophobic interactions are mainly responsible for the higher partition coefficients of cytochrome-c in aqueous two-phase systems at pH 14, and the extraction and separation capacity of biomolecules can be improved by the modulation of the relative hydrophobicity of the phases and/or the pH of the system.

Topics: Amino Acids; Anions; Cytochromes c; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Imidazoles; Ionic Liquids; Liquid-Liquid Extraction; Models, Chemical; Phosphates; Potassium Compounds; Regression Analysis; Solubility; Thermodynamics