Purification of .alpha.N-acetylgalactosaminidase

A method for purifying .alpha.-N-acetylgalactosaminidase enzyme from avian liver consisting essentially of the steps of:

• (a) homogenizing fresh or frozen-thawed avian liver in a buffer having a pH of about 3.0-4.0 and comprising acetate, citrate, phosphate or phosphate-citrate, centrifuging said homogenate to obtain a supernatant, and decanting the supernatant;
• (b) adding ammonium sulfate to the decanted supernatant to a saturation of about 30% to form a first precipitate containing contaminating proteins, removing the first precipitate, adding ammonium sulfate to the supernatant to a saturation of about 50-70% to obtain a second precipitate comprising the enzyme, and recovering the second precipitate;
• (c) dissolving the second precipitate in a buffer having a pH of about 4.5-5.5 and comprising acetate, phosphate, phosphate-citrate, citrate or Tris-HCl, and dialyzing against the same buffer;
• (d) applying the dialyzed precipitate to a cation exchange column;
• (e) washing the cation exchange column with a buffer having a pH of about 4.5-5.5 and comprising acetate, phosphate, phosphate-citrate, citrate or Tris-HCl at a concentration of about 0.05M, eluting the enzyme-containing fraction with a buffer having a pH of about 4.5-5.5 and comprising acetate, phosphate, phosphate-citrate, citrate or Tris-HCl at a concentration of about 0.25M, and concentrating and dialyzing the eluted enzyme-containing fraction;
• (f) applying the dialyzed enzyme-containing fraction to a column containing an .epsilon.-aminocaproylgalactosylamine agarose affinity resin;
• (g) washing the affinity resin with a buffer having a pH of about 4.0-5.5 and a salt concentration of about 0.25M, eluting the enzyme with a buffer having a pH of about 4.5-5.5 and comprising at least 50 mM N-acetylgalactosamine, and concentrating and equilibrating the eluted enzyme;
• and
• (h) subjecting the concentrated, eluted enzyme to gel filtration or molecular exclusion chromatography with a resin having a molecular weight cutoff of approximately 100,000 daltons, and recovering the enzyme subjected thereto.