Purification and characterization of β-N-acetylhexosaminidase I2 from human liver

Abstract

β-N-acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-β-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 μmol/min per mg of protein compared with values of 150 and 320 μmol/min/mg of protein for β-N-acetylhexosaminidases A and B purified from the same tissue. K(m) values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an M(r) between 100000 and 110000. β-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of M(r) 56000 and 29000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase α-subunit) serum confirmed that the 56000-M(r) component was the α-subunit and anti-(hexosaminidase B) serum reacted with the 29000 M(r) component. β-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.