Previous studies of the subunit structure of hexosaminidase gave ambiguous results, but suggested that the enzyme was composed of six equally sized subunits. Dissociation of hexosaminidase A with p-chloromercuribenzoate produces an alkylated fragment with mol.wt. approx. 50000, which is converted into hexosaminidase S by treatment with dithiothreitol. Treatment of native hexosaminidase A with sodium dodecylsulphate results in the formation of a large and a small fragment. However, although the native enzyme has a sedimentation coefficient of 5.8S, dissociation by S-carboxymethylation and maleic anhydride treatment results in subunits exhibiting a single schlieren boundary on analytical ultracentrifugation with a sedimentation coefficient of 2.18S. These results indicate that the enzyme is composed of four subunits, each with molwt. approx. 25000-27000. The mol.wt. of the native enzymes is calculated to be approx. 110000. Our data are consistent with the subunit structures of hexosaminidases A, B and S as being alpha2beta2, beta4 and alpha4 respectively.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.