[28] Galactose oxidase from commercial samples

Publisher This chapter describes a simple affinity procedure that removes contaminants from galactose oxidase. It provides the details concerning the stability and storage of the enzyme, the measurements of enzymic activity, and its purity. Agarose is a polysaccharide consisting of an alternating sequence of D-galactose and 3,6-anhydro-L-galactose with β(1 → 4) and α(1 → 3) links. During column chromatography at room temperature, galactose oxidase partially recognizes agarose as a galactose substrate and binds weakly to the polysaccharide. Consequently, galactose oxidase is retarded by the column and emerges well separated from the contaminating proteins, which have no affinity for the matrix. The agarose affinity procedure is a simple method for purifying an impure sample of galactose oxidase. The isolated enzyme is very stable if stored frozen in 0.05 M phosphate buffer, pH 6.5, at low temperature. Sepharose chromatography offers an effective means to separate an oxidized galactose-terminating product from galactose oxidase after reaction.