Purification and characterization of glycolic acid oxidase from pig liver

Abstract

1. 1. A procedure for the isolation of glycolic acid oxidase (glycolate:O2 oxidoreductase, EC 1.1.3.1) from pig liver is described. The enzyme has been crystallized and the amino acid composition has been determined.2. 2. Glycolic acid oxidase binds a variety of anions. Sulfate, sulfite, chloride, heptanoate, oxalate and phosphate each cause characteristic changes in the visible absorption spectrum of the enzyme. Studies on these effects have suggested that the FMN prosthetic group may be near one or more positively charged groups and also a hydrophobic region of the protein.3. 3. The pK for the ionization of the 3-imino nitrogen in free FMN (pK = 10.3) is shifted to about pH 8 in FMN bound to glycolic acid oxidase. The observed pK is increased (to pH 9.3) when the enzyme titration is performed in the presence of oxalate.4. 4. Glycolic acid oxidase contains a second chromophore which has not yet been identified. FMN can be selectively removed from the protein to give a flavin-free protein which is green in color. This green material has absorption maxima at 328,425, and 600 m[mu] and a fluorescence emission maximum at about 450 m[mu].5. 5. The visible absorption of glycolic acid oxidase is bleached in an autogenous reaction. This reaction, which is spectrally similar to the bleaching that occurs when sulfite is added to the enzyme, has been partly characterized.6. 6. The unusual absorption spectrum of pig liver glycolic acid oxidase and possibly other glycolic acid oxidases is due, at least in part, to anion binding, the presence of the unidentified green chromophore, and autogenous bleaching.