CAI Yan,HAN Peng-cheng,QI Mei-yu,et al.Analysis of Higher Structure Changes of Soybean Lipoxygenase-1 During Thermal Inactivation[J].Soybean Science,2021,40(05):703-707.[doi:10.11861/j.issn.1000-9841.2021.05.0703]
大豆脂肪氧合酶LOX-1热失活的高级结构变化分析
- Title:
- Analysis of Higher Structure Changes of Soybean Lipoxygenase-1 During Thermal Inactivation
- Keywords:
- Soybean; Lipoxygenase; LOX-1; Inactivation kinetics; FTIR; Secondary structure
- 文献标志码:
- A
- 摘要:
- 为促进大豆脂肪氧合酶LOX-1的动力学及催化机理研究，并为LOX-1活性的合理调控提供科学依据，本研究利用FT-IR技术分析LOX-1在20～80 ℃范围内的热失活和高级结构变化，采用Omnic 6.0和Peakfit 4.12软件处理FT-IR光谱，结合傅里叶自去卷积(FSD)、二阶导数分辨和曲线拟合技术分析酰胺I带，获得不同温度下LOX-1的二级结构含量，并进行热失活条件下LOX-1荧光光谱分析。研究结果显示：LOX酶活在50 ℃和55 ℃下随时间逐步降低，65 ℃时3 min内活性完全丧失，LOX-1酶对热具有敏感性。50～65 ℃时，α-螺旋和β-折叠的含量均在25%左右浮动，变化幅度较热失活小，说明LOX-1二级结构的改变不是其活性丧失的主要原因。荧光光谱显示时间延长和温度升高后，LOX-1的荧光发射峰未发生明显改变，说明50～65 ℃时活性位没有进一步将色氨酸残基暴露于溶剂中，很可能活性位只发生了微小变化。
- Abstract:
- In order to provide a new perspective for the study of LOX-1 protein tissue，promote the study of its kinetics and catalytic mechanism，and provide a useful reference for the rational regulation of LOX-1 activity,the thermal inactivation and secondary structure change of LOX-1 in the temperature range of 20-80 ℃ were studied by FT-IR.The FT-IR spectra were processed by Omnic 6.0 and Peakfit 4.12 software，and the amide I band was analyzed by Fourier self deconvolution(FSD)，second derivative resolution and curve fitting techniques.The secondary structure content of LOX-1 at different temperatures were obtained，and the fluorescence spectra under thermal inactivation conditions were analyzed.The results showed that LOX-1 activity decreased gradually with time at 50 ℃ and 55 ℃，and completely lost within 3 min at 65 ℃.LOX-1 was sensitive to heat.During 50-65 ℃，the content of α-helix and β-sheet fluctuated about 25%，and the change range was not as large as that of thermal inactivation，which indicated that the change of LOX-1 secondary structure was not the main reason for the loss of LOX-1 activity.The fluorescence spectrum showed that the fluorescence emission peak of LOX-1 did not change significantly with the increase of time and temperature，indicating that the tryptophan residues were not further exposed to the solvent，so it is likely that the active site only changed slightly.
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备注/Memo
收稿日期：2021-03-31