Haq Soghra Khatun, Ahmad Md Faiz, Khan Rizwan Hasan
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India.
Biochem Biophys Res Commun. 2003 Apr 4;303(2):685-92. doi: 10.1016/s0006-291x(03)00383-8.
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.
利用稳态色氨酸荧光、圆二色性（CD）和ANS（1-苯胺基-8-萘磺酸）结合，对黑曲霉葡萄糖氧化酶（β-D-葡萄糖：氧1-氧化还原酶）（GOD）的酸诱导去折叠进行了系统研究。内在色氨酸荧光研究表明，在pH 2.6时处于最大去折叠状态，在pH 1.4附近存在非天然中间体。黄素腺嘌呤二核苷酸（FAD）荧光测量表明，结合的辅因子在低pH下释放。在所研究的pH范围内，近紫外和远紫外CD光谱显示，在pH 2.6时三级结构和二级结构的损失最大（40%），尽管与6M盐酸胍中的构象相比，该pH下的葡萄糖氧化酶变性相对较小。有趣的是，在pH 1.4附近，葡萄糖氧化酶呈现出重新折叠的构象（A态），具有约90%的天然二级结构和类似天然的近紫外CD光谱特征。然而，ANS荧光研究表明，在pH 1.4时染料与蛋白质的结合最大，表明存在“熔球”样构象，疏水表面积增加。丙烯酰胺猝灭数据显示猝灭剂对色氨酸的可及性降低，表明在低pH下构象更紧凑。通过相对于天然蛋白质在222 nm处的椭圆率变化评估了该状态的热稳定性。虽然天然葡萄糖氧化酶显示出完全可逆的热变性曲线，但pH 1.4时的状态显示约50%的结构损失，变性状态似乎处于不同的构象，表现出突出的β-折叠结构（约85℃），且不可逆。总之，GOD的A态以具有“熔球”样特征的紧密折叠中间体形式存在，即具有类似天然的二级结构，但辅因子环境非天然，疏水表面积增加且热去折叠不协同。本研究中一个有趣的发现是，A态也具有显著的三级结构。
