Purification and some properties of bovine liver .BETA.-acetylhexosaminidase.

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Abstract
Three affinity adsorbents, p-aminophenyl .beta.-acetylglucosaminide, p-aminophenyl .beta.-1-thio-acetylglucosaminide and .beta.-acetylglucosaminylamine, each bound to CH-Sepharose 4B (GO, GS and GN, respectively), were examined for the purification of bovine liver .beta.-acetylhexosaminidases [EC 3.2.1.30]. In preliminary experiments with crude enzyme, columns of GO and GS gave 3 fractions when eluted successively with 0.05 M citrate buffer, pH 5.0, 0.1 M borate buffer, pH 10, containing 1 M sodium chloride and 5 M urea. Purification procedures involve ammonium sulfate precipitation, treatment at pH 3.8 at 37.degree., 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 MW of the hexosaminidase A and B were estimated to be 280,000, and 320,000, respectively, as determined by gel filtration using the partially purified enzymes.