Studies on Human β-d-N-Acetylhexosaminidases: I. PURIFICATION AND PROPERTIES

Abstract

Abstract β-d-N-Acetylhexosaminidase A and B were purified from human placenta. In the initial steps of purification, namely, extraction, ammonium sulfate precipitation, lyophilization, Sephadex G-200 filtration, and DEAE-cellulose column chromatography hexosaminidase A and hexosaminidase B were purified together. Hexosaminidase A was further purified by a second DEAE-cellulose column chromatography followed by ECTEOLA-cellulose column chromatography, Sephadex G-200 filtration, electrofocusing, and a third Sephadex G-200 filtration. Hexosaminidase B was further purified by CM-cellulose column chromatography, electrofocusing, and Sephadex-G-200 filtration. After the first Sephadex G-200 filtration hexosaminidase A and B were also purified by a second method. This method involved calcium phosphate gel, DEAE-Sephadex, ECTEOLA-cellulose, CM-Sephadex and CM-cellulose column chromatography, and preparative polyacrylamide disc electrophoresis. Purified hexosaminidase A and hexosaminidase B moved on polyacrylamide disc electrophoresis as single protein bands with virtually all of the enzyme activity. Hexosaminidase A and B in 10 mm phosphate buffer containing 0.1 m (NH4)2SO4 were stable at 4° for at least 4 months. The isoelectric points of hexosaminidase A and hexosaminidase B were found to be pH 5.4 and 7.9, respectively. Placental hexosaminidases were found to differ electrophoretically and antigenically from the liver and fibroblast enzymes. In contrast to the reports of others neuraminidase failed to convert heat-labile hexosaminidase A to a heatstable form of enzyme resembling hexosaminidase B.