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

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Abstract
.beta.-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-.beta.-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 .mu.mol/min per mg of protein compared with values of 150 and 320 .mu.mol/min per mg of protein for .beta.-N-acetylhexosaminidases A and B purified from the same tissue. Km 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 Mr between 100,000 and 110,000. .beta.-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56,000 and 29,000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase .alpha.-subunit) serum confirmed that the 56,000-Mr component was the .alpha.-subunit and anti-(hexosaminidse B) serum reacted with the 29,000 Mr component. .beta.-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.