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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
Author NameAffiliationE-mail
Xiao Hourong Department of Chemistry, University of Science and Technology of China, Hefei 230026  
Zhu Renfa Department of Chemistry, University of Science and Technology of China, Hefei 230026  
Xu Xiaolong Department of Chemistry, University of Science and Technology of China, Hefei 230026  
Xie Yongshu Department of Chemistry, University of Science and Technology of China, Hefei 230026  
Liu gingliang Department of Chemistry, University of Science and Technology of China, Hefei 230026 qliu@ustc.edu.cn  
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:
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*
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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