title: The acid-induced state of glucose oxidase exists as a compact folded intermediate.
abstract: A systematic investigation of the acid-induced unfolding of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase) (GOD) from Aspergillus niger was made using steady-state tryptophan fluorescence, circular dichroism (CD), and ANS (1-anilino 8-naphthalene sulfonic acid) binding. Intrinsic tryptophan fluorescence studies showed a maximally unfolded state at pH 2.6 and the presence of a non-native intermediate in the vicinity of pH 1.4. Flavin adenine dinucleotide (FAD) fluorescence measurements indicate that the bound cofactors are released at low pH. In the pH range studied, near- and far-UV CD spectra show maximal loss of tertiary as well as secondary structure (40%) at pH 2.6 although glucose oxidase at this pH is relatively less denatured as compared to the conformation in 6M GdnHCl. Interestingly, in the vicinity of pH 1.4, glucose oxidase shows a refolded conformation (A-state) with approximately 90% of native secondary structure and native-like near-UV CD spectral features. ANS fluorescence studies, however, show maximal binding of the dye to the protein at pH 1.4, indicating a "molten-globule"-like conformation with enhanced exposure of hydrophobic surface area. Acrylamide quenching data exhibit reduced accessibility of quencher to tryptophan, suggesting a more compact conformation at low pH. Thermal stability of this state was assessed by ellipticity changes at 222 nm relative to native protein. While native glucose oxidase showed a completely reversible thermal denaturation profile, the state at pH 1.4 showed approximately 50% structural loss and the denatured state appeared to be in a different conformation exhibiting prominent beta-sheet structure (around 85 degrees C) that was not reversible. To summarize; the A-state of GOD exists as a compact folded intermediate with "molten-globule"-like characteristics, viz., native-like secondary structure but with non-native cofactor environment, enhanced hydrophobic surface area and non-cooperative thermal unfolding. That the A-state also possesses significant tertiary structure is an interesting observation made in this study.
authors.affiliation: Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India.
authors.forename: Soghra Khatun
authors.initials: SK
authors.lastname: Haq
authors.forename: Md Faiz
authors.initials: MF
authors.lastname: Ahmad
authors.forename: Rizwan Hasan
authors.initials: RH
authors.lastname: Khan
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology; D002942:Circular Dichroism; D005949:Glucose Oxidase / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005949:Glucose Oxidase