Purification of a β-d-galactosidase from bovine liver by affinity chromatography

A β-d-galactosidase from bovine liver was purified to apparent homogeneity. The major purification step was affinity chromatography on a column of d-galactose attached to a Sepharose support activated with divinyl sulfone. Affinity media prepared by binding ligands to Sepharose activated with cyanogen bromide were unsuitable for purification of the enzyme, even though such media have been used to purity β-d-galactosidases from other sources. The molecular weight of the denatured enzyme was 67,000. The molecular weight of the native enzyme at pH 7.0 was 68,000, and at pH 4.5 or 5.0, was 141,000. These data suggest that the enzyme has a single, fundamental subunit with a molecular weight of 67,000, and that the enzyme exists as a monomer at pH 7.0, and a dimer at pH 4.5 or 5.0. The V_max values of the enzyme with p-nitrophenyl β-d-galactoside, p-nitrophenyl β-d-fucoside, lactose, and β-Gal-(1→4)-β-GlcNAc-1→OC₆H₄NO₂-p were 10,204, 11,550, 9,479, and 8,859 nmol/min/mg of protein, respectively, and the K_m values for these substrates were 0.08, 14.9, 14.2, and 1.6mm, respectively. d-Galactose, β-d-galactosylamine, p-aminophenyl 1-thio-β-d-galactoside, and d-galactono-1,4-lactone were competitive inhibitors of the enzyme, with K_i values of 0.9, 0.6, 0.6, and 0.8mm, respectively. The enzyme catalyzed the transfer of the d-galactosyl group from p-nitrophenyl β-d-galactoside to d-glucose. The pH optimum of the enzyme was 4.5, and the pI was 4.7.