Identification of the factor responsible for autogenous bleaching of glycollate oxidase.
Autogenous bleaching of glycollate oxidase (glycollate:oxygen oxidore-ductase, EC 1.1.3.1) is characterized by a loss of absorption due to enzyme-bound FMN and the presence of a lag in the catalytic assay. The extent of bleaching varies with different preparations. The following evidence indicates that bleaching is due to the presence of sulfite in the enzyme preparation which forms a reversible complex (EFMN + Y = EFMN . Y, Y = sulfite) with enzyme-bound FMN. Both EFMN . Y and authentic enzyme-sulfite complex exhibit similar spectral and catalytic properties. At pH 7.0 both complexes are decomposed by agents (2,6-dichlorophenolindophenol, H2O2) which cause sulfite oxidation. In the absence of such agents the complexes are extremely stable at pH 7.0 even during extensive dialysis. A pronounced decrease in complex stability is observed at pH 9.0 and 25 degrees C. Both sulfite and Y are readily removed by dialysis under these conditions. At lower temperatures both complexes exhibit an increase in stability at pH 9.0. Reversible association-dissociation is observed with EFMN . Y and EFMN . SO3 at pH 9.0, when the temperature is varied between 25 and 0 degrees C. The supernatant obtained after heat denaturation of EFMN . Y causes bleaching when mixed with EFMN, indicating that Y is present in the heat extract. A stable Bunte salt is formed by reacting sulfite with 4,4'-dithiodipyridine. Reaction of the latter with the heat extract yields a product with spectral and chromatographic properties identical to the authentic Bunte salt, indicating that the heat extract contains sulfite.
D015086:2,6-Dichloroindophenol; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D005486:Flavin Mononucleotide / Q000378:metabolism*; D006861:Hydrogen Peroxide; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013447:Sulfites; D013552:Swine
D013447:Sulfites; D005486:Flavin Mononucleotide; D006861:Hydrogen Peroxide; D015086:2,6-Dichloroindophenol; D000429:Alcohol Oxidoreductases; C020821:glycollate oxidase