Yozo Kabasawa

Protein Separation by Improved Cross-Axis Coil Planet Centrifuge with Eccentric Coil Assemblies

The new prototype of the cross-axis coil planet centrifuge (X-axis CPC) fabricated in our laboratory provides various improvements over the original unit such as ambient temperature control, good visibility of the rotary frame and substantial reduction of the torque by a round transparent case, direct motor shaft coupling to the rotary frame to stabilize the system, and ease of belt tension adjustment using idler pulleys. The capability of the system was demonstrated in the separation of stable proteins with a polymer phase system using a pair of eccentric coil assembly separation columns. Cytochrome C, myoglobin and trypsinogen were well resolved and eluted in 5.5 h at a partition efficiency of 200 theoretical plates. The method provides a gentle environment for proteins without causing their deactivation or loss.

Resolution of DL-Valine by Countercurrent Solvent Extraction with Continuous Sample Feeding

Abstract DL-Valine was resolved completely by a solvent extraction system with a continuous sample feeding process called continuous countercurrent fractional extraction (CFE). The two-phase system contained n-butanol and water with a copper(II) complex of N-n-dodecyl-L-hydroxproline as a resolving reagent. The upper (alcohol-rich) phase and the lower (water-rich) phase proceeded in the segmented extraction columns countercurrently; the D-isomer was recovered in the upper phase, while the L-isomer was recovered in the lower phase. The optical purity of the enantiomers obtained was consistently 99.5% or higher. The D-isomer extracted in the upper phase was recovered completely by using a backextraction column in series after the extraction column, allowing the upper phase to be used repeatedly for the resolution. The efficiency of the CFE system was estimated at more than 24 theoretical stages.

Protein separation by nonsynchronous coil planet centrifuge with aqueous–aqueous polymer phase systems

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.

Countercurrent chromatographic analysis of ovalbumin obtained from various sources using the cross-axis coil planet centrifuge.

The present studies have been conducted to investigate the cause of an unusually broad peak of ovalbumin obtained by countercurrent chromatography (CCC) reported earlier [K. Shinomija et al., J. Chromatogr., 644 (1993) 215]. A series of CCC experiments using our prototyte of the cross-axis coil planet centrifuge revealed that commercial ovalbumin products were classified into two groups: group A formed two peaks of ovalbumin at pH 7.0 and 5.8, while group B showed a relatively sharp single peak in a broad range of pH. Electrophoresis indicated that the group A ovalbumin consisted of both natural and denatured products: the natural ovalbumin is a monomer (Mr 45 000) whereas the denatured products form dimers (Mr 90 000). The abnormally broad peak obtained from the group A ovalbumin at pH 9 is apparently caused by the heterogeneity of the sample protein.

Enantiomeric Separation of Commercial D,L-Kynurenine with an Aqueous Two-Phase Solvent System by Cross-Axis Coil Planet Centrifuge

Commercial D,L-kynurenine was resolved by high-speed countercurrent chromatography using a cross-axis coil planet centrifuge. The separation was performed with an aqueous-aqueous polymer phase system composed of 10% (w/w) polyethylene glycol 8000 and 5% (w/w) dibasic sodium phosphate containing 6% (w/w) bovine serum albumin as a chiral selector. The lower phosphate-rich mobile phase eluted the L-enantiomer first followed by the D-enantiomer. The peak resolution was 0.94 for 2.5 mg of the sample. The separation was completed within 3.5 h.

COUNTERCURRENT CHROMATOGRAPHIC SEPARATION OF SUGARS AND THEIR p-NITROPHENYL DERIVATIVES BY CROSS-AXIS COIL PLANET CENTRIFUGE

Sugars and their p-nitrophenyl (PNP) derivatives were separated by high-speed countercurrent chromatography using a cross-axis coil planet centrifuge (cross-axis CPC) equipped with a pair of eccentric coil assemblies. A polar two-phase solvent system composed of 1-butanol/acetic acid/water (4:1:5) was used for the separation of sucrose and fucose, and glucuronic acid (lactoid form) and galacturonic acid, while 1-butanol/ethanol/water (4:1:4) was used for the separation of free and lactoid forms of glucuronic acid. PNP-sugar derivatives such as neutral sugars, uronic acids, and amino sugars were separated with a less polar solvent system composed of n-hexane/ethyl acetate/1-butanol/methanol/water at various volume ratios. PNP-glucose derivatives were further separated according to the number of sugar chains, and five PNP-neutral sugars were resolved by adding 0.1 M sodium tetraborate in the two-phase solvent system. Overall results of experiments revealed that the cross-axis CPC is useful for the separatio...

Protein Separation by Cross-Axis Coil Planet Centrifuge with Two Different Types of Coiled Columns

Effect of elution modes on protein separation was investigated using cross-axis coil planet centrifuge (cross-axis CPC) with two different types of coiled columns, i.e., eccentric coil and toroidal coil assemblies. Myoglobin and lysozyme were separated with an aqueous two-phase solvent system composed of 12.5% (w/w) polyethylene glycol 1000 and 12.5% (w/w) dibasic potassium phosphate. The substantial effect of elution modes was observed by the toroidal coil, while the negative result was given at the eccentric coil. Using the toroidal coil, higher peak resolution of proteins was attained at the tail to head elution mode. In the outward lower phase mobile elution mode, the satisfactory separation was obtained by both eccentric coil and toroidal coil assemblies. However, in the inward upper phase mobile elution mode, the toroidal coil produced a broad and asymmetric myoglobin peak. The analysis using polyacrylamide gel electrophoresis revealed that the toroidal coil partially separated the components originally present in the myoglobin sample. As the result, a modified equation was devised to express the peak resolution (Rs) using the lysozyme peak. The overall results indicated that the toroidal coil produced better partition efficiency than the eccentric coil under the optimized experimental condition including the direction of the Coriolis force acting on the mobile phase in the toroidal coil.

Effect of Elution Modes on Protein Separation by Cross-Axis Coil Planet Centrifuge with Two Different Types of Coiled Columns

ABSTRACT Effect of elution modes on protein separation was investigated using cross-axis coil planet centrifuge (cross-axis CPC) with two different types of coiled columns, i.e., eccentric coil and toroidal coil assemblies. Myoglobin and lysozyme were separated with an aqueous two-phase solvent system composed of 12·5% (w/w) polyethylene glycol 1000 and 12·5% (w/w) dibasic potassium phosphate. The substantial effect of elution modes was observed by the toroidal coil, while the negative result was given at the eccentric coil. Using the toroidal coil, higher peak resolution of proteins was attained at the tail to head elution mode. In the outward lower phase mobile elution mode, the satisfactory separation was obtained by both eccentric coil and toroidal coil assemblies. However, in the inward upper phase mobile elution mode, the toroidal coil produced a broad and asymmetric myoglobin peak. The analysis using polyacrylamide gel electrophoresis revealed that the toroidal coil partially separated the componen...

Time-resolved fluorescence receptor assay for benzodiazepines

Abstract A novel non-isotopic receptor binding technique for the detection of benzodiazepines is described. A benzodiazepine labeled with europium chelate was prepared and employed as a labeled ligand, and time-resolved measurements of the long lifetime fluorescence of europium chelate allowed avoidance of interference due to proteins in the receptor preparation. Experimental results demonstrate a sigmoid inhibition curve, with binding of the labeled ligand inhibited by comparable concentrations of the unlabeled drug. The proposed assay may provide a simple procedure for the measurement of benzodiazepines in biological systems and a useful tool in the screening of natural substances for new classes of benzodiazepine-like compounds.

PROTEIN SEPARATION BY CROSS-AXIS COIL PLANET CENTRIFUGE WITH SPIRAL COLUMN ASSEMBLIES

ABSTRACT A newly fabricated spiral column assembly was first applied to the countercurrent chromatographic separation of proteins using the cross-axis coil planet centrifuge (cross-axis CPC). The separation was performed by a set of stable proteins such as cytochrome C, myoglobin, and lysozyme with an aqueous–aqueous polymer phase solvent system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000–12.5% (w/w) dibasic potassium phosphate. Three sets of left-handed single-layer spiral columns with different IDs (1.0, 1.5, and 2.0 mm) were employed to investigate the effect of column ID on partition efficiency at four different elution modes. The elution modes consisted of the combinations of the direction of revolution (PI=counterclock-wise; PII=clockwise), the head–tail elution mode (H=head to tail; T=tail to head), and the inward–outward elution mode (I=inward; O=outward). Among these experiments, the best separation of proteins was attained using a 1.5 mm-ID column assembly with the PI-H-O elution mode of the lower phase mobile. The resolution between cytochrome C and myoglobin peaks was 0.9 and between myoglobin and lysozyme peaks was 1.0, while the retention of the stationary phase was 32.7%. In order to improve the partition efficiency, the four-layer spiral column assembly with 1.0 mm-ID column tubing was applied to the protein separation. The resolution between these peaks was improved to 1.1 and 1.2, respectively, while the stationary phase retention decreased to 20.9%. Further studies using a single-layer spiral coiled column also revealed the effective peak resolution of proteins, but with lower stationary phase retention. The overall results demonstrated the spiral column assembly was useful for the protein separation using the cross-axis CPC with the aqueous-aqueous polymer phase system.