Hiroo Sasaki

Separation range and separation efficiency in high-speed gel filtration on TSK-GEL SW columns

Abstract Separation ranges for gel filtration on TSK-GEL SW columns, G2000SW, G3000SW and G4000SW, were measured for globular protein, dextran and polyethylene glycol. It was possible to separate globular proteins of molecular weights from 5000 to 7,000,000, dextrans of molecular weights from 1000 to 500,000 and polyethylene glycols of molecular weights from 500 to 250,000 by using these three columns of different pore sizes. The column having the highest separation efficiency for globular proteins was also determined. The highest separation efficiency for molecular weights of less than 30,000, 30,000–500,000 and greater than 500,000 was exhibited by G2000SW, G3000SW and G4000SW, respectively.

Evaluation of new supports for high-pressure aqueous gel permeation chromatography: TSK-GEL SW type columns

Abstract New chemically modified silica gel based supports, TSK-GEL SW type columns, for aqueous gel permeation chromatography have been evaluated in detail. Adsorption or denaturation of proteins and enzymes on these supports is negligible, the recoveries of the proteins and enzyme activities being both almost quantitative. The applications of these supports for the study of proteins, enzymes, saccharides and water-soluble sythetic polymer have been investigated.

High-speed gel filtration of proteins in sodium dodecyl sulphate aqueous solution on TSK-GEL SW type

High-speed gel filtration of proteins was performed in sodium dodecyl sulphate aqueous solutions on TSK-GEL SW type columns. The separation ranges of G2000SW, G3000SW and G4000SW were, respectively, 15,000–25,000, 10,000–100,000 and 15,000–300,000. G3000SW showed the highest separation efficiency in the molecular weight range below ca. 60,000, while G4000SW showed the highest separation efficiency above molecular weight of ca. 60,000. The elution behaviour of proteins was greatly affected by sodium phosphate concentration in the eluent and the optimum sodium phosphate concentration was 0.05–0.2 M.

Separation of proteins by hydrophobic interaction chromatography at low salt concentration.

We investigated protein separation by hydrophobic interaction chromatography (HIC) at low salt concentration on the supports of various hydrophobicities. Hydrophobic proteins could be successfully separated with more than 90% recovery by gradient elution of ammonium sulfate from 0.3-0.5 M to 0 in 50 mM phosphate buffer (pH 6.8) by using supports whose hydrophobicities were properly adjusted individually for each protein. Satisfactory results were also obtained by isocratic elution without ammonium sulfate and gradient elution of ethanol from 0 to 10%. HIC at low salt concentration was compatible with other modes of liquid chromatography like ion-exchange chromatography. On the other hand, it was not successful to separate hydrophilic proteins at low salt concentration. Recoveries of hydrophilic proteins decreased before they were retained enough as support hydrophobicity increased. Therefore, it is inevitable to use a higher concentration of salt, e.g., 1-2 M ammonium sulfate, on hydrophilic or moderately hydrophobic support in order to retain hydrophilic proteins without decrease in recovery.

Purification of enzymes by high-speed gel filtration on TSK-GEL SW columns

Abstract The purification of enzymes was investigated by high-speed gel filtration on TSK-GEL G3000SWG columns packed with porous silica gel deactivated by chemically bonded hydrophilic compounds. Crude β-galactosidase from bacterial cells and commercial urease were purified ca. 15-fold in a single gel filtration. These enzymes were eluted within an hour from the column and the recoveries of enzymatic activity were almost 100% although the operation was carried out at room temperature (22°). Samples up to 100 mg could be applied to the column without loss of separation efficiency.

Characterization of p-aminobenzamidine-based sorbent and its use for high-performance affinity chromatography of trypsin-like proteases

An affinity sorbent, hydrophilic polymer-based carrier of different pore size (Toyopearl) with immobilized p-aminobenzamidine (ABA), has been prepared. Its basic properties and some applications for protein purification were studied. ABA, which is a synthetic inhibitor for trypsin-like proteases, was covalently immobilized to Toyopearl by reductive amination. The ligand density and binding capacity for porcine trypsin varied depending on the pore size of Toyopearl. The maximum binding capacity of the immobilized p-aminobenzamidine Toyopearl (ABA-Toyopearl) for trypsin was more than 40 mg/ml gel. ABA-Toyopearl thus obtained was very stable below pH 8 and was successfully used for high-performance affinity chromatography of trypsin-like proteases such as trypsin, thrombin, tissue-type plasminogen activator or urokinase in a single step at 25 degrees C.

Ion-exchange high-performance liquid chromatographic studies on sulphydryl-catalysed structural alterations of bovine mercaptalbumin

The N-A isomerization (the intramolecular SH/S-S exchange reaction) of bovine mercaptalbumin (BMA) in alkaline medium was studied by using ion-exchange high-performance liquid chromatography (HPLC) and moving-boundary electrophoresis. Results obtained by ion-exchange HPLC on the N-A isomerization of BMA were consistent with those by moving-boundary electrophoresis and showed at least two kinds of the A-form, A1 and A2, indicating that the N-A isomerization is a multi-step reaction. The rate of the N-A isomerization was strongly suppressed in [2H]water solution. The suppression by [2H]water might support the current view that intra- and intermolecular hydrophobic and/or hydrogen bonds are strengthened in [2H]water.

Hydrophobicity gradient columns for the separation of trypsin inhibitor by hydrophobic interaction chromatography at low salt concentration

We investigated hydrophobicity gradient columns composed of two columns packed with supports of different hydrophobicities in order to save time in protein separation by hydrophobic interaction chromatography at low salt concentration using a crude sample of trypsin inhibitor as a model sample. One of the two hydrophobicity gradient columns was packed with a support whose hydrophobicity was critically controlled for target protein (trypsin inhibitor) and the other was packed with a support which was less hydrophobic than the critically controlled hydrophobicity support. It was found that the hydrophobicity gradient columns are useful to separate samples containing impurities of a wide range of hydrophobicities within a reasonable time.

Evaluation and applications of a new dye affinity adsorbent.

The basic properties of a new dye affinity adsorbent Toyopearl AF-Blue HC-650M and its applications to the purification of proteins were studied. The binding capacity for human serum albumin (HSA) was greater than 18 mg per ml gel. The dye leakage from Toyopearl AF-Blue HC-650M in 0.5 M NaOH and 0.5 M HCI was less compared with an agarose adsorbent. Caustic stability study also demonstrated this material withstood exposure to 0.1 M NaOH for 1 month with no significant loss of binding capacity for HSA. We purified human albumin from human serum and lactate dehydrogenase (LDH) from rabbit muscle extract in a single step. Sodium dodecylsulfate-polyacrylamide gel electrophoresis indicates that human albumin and LDH were highly purified.

Effect of chromatographic conditions on resolution in high-performance ion-exchange chromatography of proteins on nonporous support

We explored chromatographic conditions to obtain high resolution in protein separations by ion-exchange chromatography (IEC) on a nonporous anion-exchange resin of 2.5 microm in particle diameter. We studied the effects of gradient time (steepness of salt concentration gradient), flow-rate and column length on resolution in much wider ranges than had been studied before. It was found that two distinct conditions exist that provide high resolution. The first is a condition which has widely been employed in current high-performance IEC, namely, a combination of short gradient time, high flow-rate and comparatively short column. Separation times are usually 5-30 min, and even more rapid (1-2 min) separations are possible. The second is the condition which has rarely been employed in high-performance IEC. It is a combination of long gradient time, low flow-rate and long column. Although it takes several hours for one separation, very high resolution is attainable.