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Studies on Cu2+-induced Resistant-denaturation of PPO from Nictiana Tobacum
Xiao Hourong, Zhu Renfa, Xu Xiaolong, Xie Yongshu, Liu gingliang
Department of Chemistry, University of Science and Technology of China, Hefei 230026
Abstract:
In order to investigate the interaction between exogenous Cu2+ and Polyphenoloxidase (PPO) from Nictiana Tobacum, the Cu2+-induced resistant-denaturation against guanidine hydrochloride(Gdn-HCl) was studied by the following enzymatic activity assay, fluorescence spectroscopy and circular dichroism (CD). The results show that the GdnHCl-induced unfolding is a two-state process with no detectable inter mediate state in the absence of exogenous Cu2+, while the GdnHCl-induced unfolding in the presence of 10.0 mmol/L exogenous Cu2+ follows a three-state transition with an intermediate state, which results from the fact that Cu2+ increases the structural stability of native PPO and its intermediate. In terms of enzymatic activities, 6 mol/L GdnHCl makes PPO lose 81.4% of its original activity after 5 min, inactivate completely after 30 min, while in the presence of 10.0 mmol/L exogenous Cu2+, only 39.4% and 75.1%, after 5 and 30 min respectively. According to the CD measurements, the relative average fractions of α-helix, anti-parallel2-sheet, 2-turn/parallel 2-sheet, aromatic residues and disulfide bond, and random coil/γ-turn are 1.1%, 3.8%, 3.3%, 7.5% and 84.3%, respectively, in the 6.0mol/L GdnHCl containing no CuSO4, but34.2%, 13.7%, 21.0%, 9.5% and 21.6%, respectively, in the same concentration of Gdn-HCl containing 10mmol/L CuSO4.
Key words:  Cu2+, Polyphenol oxidase, Fluorescence spectroscopy, Circular dichroism, Unfolding
FundProject:
附件
硫酸铜诱导烟草多酚氧化酶抗变性的研究
肖厚荣, 朱仁发, 徐小龙, 解永树, 刘清亮*
中国科学技术大学化学系,合肥,230026
摘要:
为了进一步探讨外源铜与多酚氧化酶的相互作用,通过酶活性测定,荧光光谱和圆二色光谱 (CD)对外源Cu2+诱导烟草多酚氧化酶抗变性进行了研究.荧光光谱结果表明,没有外源铜存在的情况下,盐酸胍诱导的烟草多酚氧化酶变性是一个二态过程,烟草多酚氧化酶在6 mol/L盐酸胍中变性5 min,酶活性降至18.6%,变性30 min便彻底失活; 10.0 mmol/L外源铜存在时,盐酸胍诱导的烟草多酚氧化酶变性是一个三态过程,上述相同盐酸胍浓度下变性相同时间,酶的剩余活性分别为60.6%和24.9%.CD谱数据结果表明, 6 mol/L盐酸胍中变性30 min,烟草PPO二级结构中α-螺旋、反平行β-折叠、β-转角/平行β-折叠、芳香残基和二硫键、无规卷曲/γ-转角的百分含量分别为1.1、 3.8、 3.3、 7.5和84.3;在10.0 mmol/L Cu2+存在时,上述相同变性条件下,各种二级结构百分含量分别为34.2、 13.7、 21.0、 9.5和21.6.适量外源铜可明显提高烟草多酚氧化酶结构的稳定性.
关键词:  硫酸铜  多酚氧化酶  荧光光谱  圆二色光谱  变性
DOI:10.1088/1674-0068/17/5/587-591
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