Affinity Chromatography of Glycosidases

Abstract

Four adsorbents with different structures were prepared by coupling di-ε-aminocaproyl-p-aminophenyl N-acetyl-β-D-glucosaminide, β-D-glucoside, N-(di-ε-aminocaproyl)glucosamine, and N-(di-ε-aminocaproyl)glucosaminitol with CNBr-activated Sepharose 4B. Their adsorption characteristics were examined using partially purified glycosidase mixtures from Takadiastase and from the liver of abalone (Haliotis gigantea Gmelin). The elution profiles of the enzymes from these four adsorbents were very similar to each other, as well as to those from other adsorbents prepared by coupling di-ε-aminocaproyl-p-aminophenyl N-acetyl-1-thio-β-D-glucosaminide, 1-thio-β-D-glucoside, 1-thio-β-D-galactoside, and 1-thio-α-D-mannoside with CNBr-activated Sepharose 4B. The present experiments confirmed the following results (1): (A) the glycosidases were adsorbed at low ionic strength and eluted by increasing the ionic strength; (B) the glycosidases exammed could not differentiate the ligand structures in the adsorbents, notwithstanding their strict enzymatic specificity; (C) β-D-N-acetylglucosaminidases [EC 3.2.1.30] from various sources, among other glycosidases, were readily adsorbed at low ionic strength, and were eluted last by a linear gradient change of ionic strength. α-Mannosidase [EC 3.2.1.24] was eluted earlier than N-acetyl-β-glucosaminidase, but later than β-glucosithse [EC 3.2.1.21] or β-galactosidase [EC 3.2.1.23]. The following enzyme proteins, trypsin EEC 3.4.21.4], chymotrypsin [EC 3.4.21.1], lysozymes [EC 3.2.1.17] (of hen egg white, quail and duck), cytochrome c, myoglobin, glucoamylases [EC 3.2.1.3] (of Rhizopus niveus and R. genus) and amylases [EC 3.2.1.1] (of B. subtilis and hog pancreas), showed no affinity for any of these adsorbents with the exception of Taka-amylase A [EC 3.2.1.1]. Con A adsorbed on adsorbents having different glycoside ligands showed a binding specificity completely parallel to that demonstrated in the inhibition experiments of Goldstein. Con A was adsorbed so tightly on adsorbents having α-D-mannoside ligands that it could not be eluted even by 0.2 M Me-α-Man solution. Con A adsorbed on adsorbents having either β-D-glucoside or N-acetyl-β-D-glucosaminide ligands was eluted with 0.05 M Me-α-Man. The ionic strength of the solution had no effect on the binding of Con A. These results clearly show that, in this kind of affinity chromatography, there were two types of binding, one being a specific binding like that of lectin (represented by Con A) which was not affected by ionic strength; this binding was presumably due to interaction between the specific binding site of protein and the glycoside ligands in the adsorbents. The other was a non-specific binding observed with various glycosidases, which depended on ionic strength; the binding was presumably not due to interaction between the active sites of the enzymes and the glycoside ligands of the adsorbents.