Irreversible thermal denaturation of glucose oxidase from Aspergillus niger is the transition to the denatured state with residual structure.
Glucose oxidase (GOX; beta-d-glucose:oxygen oxidoreductase) from Aspergillus niger is a dimeric flavoprotein with a molecular mass of 80 kDa/monomer. Thermal denaturation of glucose oxidase has been studied by absorbance, circular dichroism spectroscopy, viscosimetry, and differential scanning calorimetry. Thermal transition of this homodimeric enzyme is irreversible and, surprisingly, independent of GOX concentration (0.2-5.1 mg/ml). It has an apparent transition temperature of 55.8 +/- 1.2 degrees C and an activation energy of approximately 280 kJ/mol, calculated from the Lumry-Eyring model. The thermally denatured state of GOX after recooling has the following characteristics. (i) It retains approximately 70% of the native secondary structure ellipticity; (ii) it has a relatively low intrinsic viscosity, 7.5 ml/g; (iii) it binds ANS; (iv) it has a low Stern-Volmer constant of tryptophan quenching; and (v) it forms defined oligomeric (dimers, trimers, tetramers) structures. It is significantly different from chemically denatured (6.67 m GdmHCl) GOX. Both the thermal and the chemical denaturation of GOX cause dissociation of the flavin cofactor; however, only the chemical denaturation is accompanied by dissociation of the homodimeric GOX into monomers. The transition temperature is independent of the protein concentration, and the properties of the thermally denatured protein indicate that thermally denatured GOX is a compact structure, a form of molten globule-like apoenzyme. GOX is thus an exceptional example of a relatively unstable mesophilic dimeric enzyme with residual structure in its thermally denatured state.
Zoldák
Gabriel
G
Department of Biochemistry, Faculty of Sciences P. J. Safárik University, Kosice, Slovakia.
Zubrik
Anton
A
Musatov
Andrej
A
Stupák
Marek
M
Sedlák
Erik
E
D001234:Aspergillus niger / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D005949:Glucose Oxidase / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D011487:Protein Conformation; D011489:Protein Denaturation
D001426:Bacterial Proteins; D005949:Glucose Oxidase