Abstract
Three affinity adsorbents, p-aminophenyl β-acetylglucosaminide, p-aminophenyl β-1-thio-acetylglucosaminide and β-acetylglucosaminylamine, each bound to CH-Sepharose 4B (GO, GS and GN, respectively), were examined for the purification of bovine liver β-acetylhexosaminidases. In preliminary experiments with crude enzyme, columns of GO and GS gave three fractions when eluted successively with 0.05M citrate buffer, pH 5.0, 0.1M borate buffer, pH 10, containing 1 M sodium chloride, and 5M urea. Purification procedures involve ammonium sulfate precipitation, treatment at pH 3.8 at 37°, Sephadex G-200 gel filtration, DEAE-cellulose column chromatography and affinity chromatography on GS. A hexosaminidase A was obtained as a electrophoretically pure protein with high specific activity, 137 units per mg. Activity in hexosaminidase B fraction showed multiplicity in its behavior in the affinity chromatography, and the high specific activity (184 units per mg) was obtained only with a GO column. Km values and ratios of acetylglucosaminidase to acetylgalactosaminidase activities were determined for main components. The molecular weights of the hexosaminidase A and B were estimated to be 280000 and 320000, respectively, as determined by gel filtration using the partially purified enzymes.
