Chromatography of beta-glucuronidase from bovine liver. A study of the enzyme binding sites of prepared adsorbents.
beta-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-beta-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 beta-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-beta-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-beta-D-glucuronic acid-CH Sepharose 4B.(ABSTRACT TRUNCATED AT 250 WORDS)
Iino
N
N
Daiichi College of Pharmaceutical Sciences, Fukuoka, Japan.
Yoshida
K
K
D000327:Adsorption; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005966:Glucuronidase / Q000737:chemistry*; D008024:Ligands; D008099:Liver / Q000201:enzymology*
D008024:Ligands; D005966:Glucuronidase