Aspergillus niger sulfhydryl oxidase.
A procedure for the isolation of a sulfhydryl oxidase from an Aspergillus niger cell suspension involved three major steps and yielded enzyme preparations exhibiting a single but diffuse protein-containing zone when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight estimated to be 53,000. Sedimentation equilibrium experiments indicated a native molecular weight of 106,000. Analyses for sugar residues showed that the enzyme is a glycoprotein, containing 20.3% neutral hexose and 1.9% aminohexose by weight. This enzyme catalyzed the conversion of reduced glutathione (GSH) to its disulfide form, with concomitant consumption of O2 and release of H2O2. The ratio of GSH consumed to H2O2 produced was determined to be 2:1. At 25 degrees C, the optimum pH for the oxidation of GSH was 5.5. Under these conditions, the enzyme had a Michaelis constant of 0.3 mM for GSH. Other low molecular weight thiol compounds (cysteine, dithiothreitol, and 2-mercaptoethanol) were also oxidized, but the Michaelis constants for these substrates were substantially higher than that for GSH under identical conditions of temperature and pH. The rate of reactivation of reductively denatured ribonuclease A was enhanced by the presence of sulfhydryl oxidase, indicating that the latter is capable of oxidizing protein-associated thiol groups. The UV-visible spectrum of sulfhydryl oxidase solution had absorbance maxima at 274, 364.5, and 442.5 nm and was otherwise characteristic of the spectra of known flavoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)
de la Motte
R S
RS
Department of Biochemistry, University of Nebraska, Lincoln 68583-0718.
Wagner
F W
FW
D000596:Amino Acids / Q000032:analysis; D001234:Aspergillus niger / Q000201:enzymology*; D002241:Carbohydrates / Q000032:analysis; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D013379:Substrate Specificity; D050862:Oxidoreductases Acting on Sulfur Group Donors
D000596:Amino Acids; D002241:Carbohydrates; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors