Purification and biochemical/biophysical characterization of two hexosaminidases from the fresh water mussel, Lamellidens corrianus

Abstract

Two thermostable isoforms of a hexosaminidase were purified to homogeneity from the soluble extract of fresh water mussel Lamellidens corrianus, employing a variety of chromatographic techniques. Hexosaminidase A (HexA) is a heterodimer with subunit masses of ~80 and 55 kDa. Hexosaminidase B (HexB) is a homodimer with a subunit mass of 55–60 kDa. Circular dichroism spectroscopic studies indicated that both HexA and HexB contain β-sheet as the major secondary structural component with considerably lower content of α-helix. The temperature and pH optima of both the isoforms were found to be 60 °C and 4.0, respectively. The IC50 values for HexA with N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, D-galactosamine, D-glucosamine, methyl α-D-mannopyranoside and D-mannose are 3.7, 72.8, 307, 216, 244 and 128 mM, respectively, whereas the corresponding IC50 values for HexB were estimated as 5.1, 61, 68, 190, 92 and 133 mM, respectively. Kinetic parameters KM and Vmax for HexA and B with p-nitrophenyl N-acetyl-β-D-glucosaminide are 4 mM, 0.23 μmol·min−1·mL−1 and 2.86 mM, 0.29 μmol·min−1·mL−1, respectively, and with p-nitrophenyl N-acetyl-β-D-galactosaminide are 4.5 mM, 0.054 μmol·min−1·mL−1 and 1.4 mM, 0.14 μmol·min−1·mL−1, respectively. GalNAc inhibited both isoforms in a non-competitive manner, whereas a mixed mode of inhibition was observed with GlcNAc with both forms.