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The acid-induced state of glucose oxidase exists as a compact folded intermediate.
Biochem Biophys Res Commun. 2003 Apr 04; 303(2):685-92.BB

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+Show Affiliations

Haq SK
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India.
Ahmad MF
No affiliation info available
Khan RH
No affiliation info available

MeSH

Aspergillus nigerCircular DichroismGlucose OxidaseHydrogen-Ion ConcentrationKineticsProtein ConformationProtein FoldingSpectrometry, FluorescenceSpectrophotometry, Ultraviolet

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

12659873

Citation

Haq, Soghra Khatun, et al. "The Acid-induced State of Glucose Oxidase Exists as a Compact Folded Intermediate." Biochemical and Biophysical Research Communications, vol. 303, no. 2, 2003, pp. 685-92.
Haq SK, Ahmad MF, Khan RH. The acid-induced state of glucose oxidase exists as a compact folded intermediate. Biochem Biophys Res Commun. 2003;303(2):685-92.
Haq, S. K., Ahmad, M. F., & Khan, R. H. (2003). The acid-induced state of glucose oxidase exists as a compact folded intermediate. Biochemical and Biophysical Research Communications, 303(2), 685-92.
Haq SK, Ahmad MF, Khan RH. The Acid-induced State of Glucose Oxidase Exists as a Compact Folded Intermediate. Biochem Biophys Res Commun. 2003 Apr 4;303(2):685-92. PubMed PMID: 12659873.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The acid-induced state of glucose oxidase exists as a compact folded intermediate. AU - Haq,Soghra Khatun, AU - Ahmad,Md Faiz, AU - Khan,Rizwan Hasan, PY - 2003/3/28/pubmed PY - 2003/6/21/medline PY - 2003/3/28/entrez SP - 685 EP - 92 JF - Biochemical and biophysical research communications JO - Biochem Biophys Res Commun VL - 303 IS - 2 N2 - 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. SN - 0006-291X UR - https://www.unboundmedicine.com/medline/citation/12659873/The_acid_induced_state_of_glucose_oxidase_exists_as_a_compact_folded_intermediate_ DB - PRIME DP - Unbound Medicine ER -