Chromatography of β-glucuronidase from bovine liver. A study of the enzyme binding sites of prepared adsorbents

Article abstract of DOI:10.1248/cpb.40.1852

β-Glucuronidase from bovine liver was adsorbed to the adsorbents prepared with CH-Sepharose 4B and either the competitive inhibitor or its analogs such as p-aminophenyl 1-thio-β-D-glucuronic acid, -glucoside, -galactoside, and N-acetyl glucosaminide. The adsorbed enzyme was eluted at 0.1 or 0.5 M NaCl by a stepwise gradient. Chromatography of the enzyme was also performed by using the adsorbents prepared with Epoxy-activated Sepharose 6B and amine compounds or other compounds. In order to see whether the hydroxyl groups of the sugar parts in the ligand are necessary for the adsorption of the enzyme, chromatography was performed by using the adsorbents prepared with sugar derivatives as the ligand. As a result, it was found that β-glucuronidase had an affinity for adsorbents prepared with either acetyl derivatives or methoxy derivatives of glycosides and CH-Sepharose 4B. From the results of elution of the enzyme with NaCl from adsorbents having amide bonding, it was clarified that the affinity of the enzyme for adsorbents without glycosides in the ligands correlated with acidity of the amide in the adsorbents. Hydrogen bond chromatography was performed with the prepared adsorbents. The enzyme was adsorbed under a high concentration of ammonium sulfate, and the elution of the adsorbed enzyme from adsorbents was examined by the degradation of salt. The enzyme was most easily eluted from aminoethyl 1-thio-β-D-glucuronic acid-CH Sepharose 4B at 0.9 M ammonium sulfate and at 0.5 m concentration of the salt with p-aminophenyl 1-thio-β-D-glucuronic acid-CH Sepharose 4B. Furthermore, the adsorbed enzyme was eluted by the addition of urea as well as ethylene glycol which are known as reagents which weaken hydrogen bonding. The results suggested that the interaction between the enzyme and the adsorbents with an amide bonding may be affected by the electrostatic force in the adsorbents under a high concentration of salt, although the electrostatic force decreases under the high concentration of salt. We also investigated whether or not the adsorbed enzyme was eluted by sodium cholate, cholic acid and triton X-100 known as hydrophobic reagents. It was assumed from the results of these chromatographies that the presence of amide bonding in adsorbents with glycosides as the ligand may be essential for the adsorption of the enzyme and that the glycosidic parts of the ligands have an effect on adsorption, however, it may not be essential for adsorption.

Full text of DOI:10.1248/cpb.40.1852

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