Purification and properties of beta-N-acetyl-D-hexosaminidase from boar seminal plasma

beta-N-Acetyl-D-hexosaminidase has been purified ca. 190-fold to homogeneity from boar seminal plasma. It catalyzed the hydrolysis of the p-nitrophenyl-N-acetyl derivatives of both beta-D-glucosaminide and beta-D-galactosaminide but was inactive with the o- or p-nitrophenyl glycosides of other monosaccharides. Its pH optimum was 4.5 and its KM was 1.5 mM with p-nitrophenyl-N-acetyl-beta-D-glucosamide as substrate. The enzyme was inhibited by mercuribenzoate compounds but not by iodoacetamide, 2,2'-dipyridyl disulfide, methylmethane thiosulfonate, nor N-ethylmaleimide. The active enzyme had mol. wt ca. 250,000 by Sephacryl S-300 chromatography. SDS electrophoresis showed single bands corresponding to subunit mol. wts ca. 62,000 and 107,000 depending on whether the enzyme had been denatured in the presence of 2-mercaptoethanol or not. These data suggest that the enzyme is a tetramer of identical subunits, pairs of which are held together by disulfide bonds.