Zn^(2+)与苹果多酚氧化酶相互作用研究

Studying on the Interaction Between Zn^(2+) and Polyphenol Oxidase from Apple

为探讨苹果多酚氧化酶(简称PPO)的结构与功能关系,应用紫外差示光谱、同步荧光光谱及圆二色性光谱(CD)等生物物理技术和酶活性测定研究了外加Zn^(2+)对苹果PPO的分子构象和酶活性的影响。结果表明,微量锌的加入能增加酶的活性和提高α-螺旋含量,当[Zn^(2+)]/[PPO]为2.0左右时,苹果PPO活性最高,继续增加[Zn^(2+)]/[PPO]比值,酶活性开始下降,比值为2.7以后,Zn^(2+)表现了对PPO活性的抑制,α-螺旋含量下降;Zn(Ⅱ)可能与PPO中的组氨酸残基进行了配位,(1mH)σN+σN→Zn^(2+)、(ImH)σ→Zn^(2+)、(ImH)_(π2)→Zn^(2+)、(ImH)_(π1)→Zn^(2+)分别为227,241,265, 335(nm);色氨酸和酪氨酸的荧光均受到Zn^(2+)的猝灭,但它们的微环境在Zn(Ⅱ)-PPO的相互作用中基本保持不变。

The effect of exo-- Zn^2＋ on the structure and activity of Polyphenol Oxidase （PPO） is studied by employing enzymatic activity assay, differential absorption spectrum, synchronous fluorescence spectra and far-- UV CD spectra, which could be used to explore the relations between their structure and function. The results show that the activity of PPO is enhanced by trial Zn^2＋ , reaching its maxium at [Zn^2＋]/[PPO] 2. 0, but inhibited when Zn^2＋ is further added. Trial Zn^2＋ can also increase and the a--helix content. Zn^2＋ may coordinate with histidine（s） in PPO, and the absorptions of （ImH）σN＋σN→Zn^2＋、（ImH）σ→Zn^2＋、（ImH）π1→Zn^2＋、（ImH）Ⅱπ→Zn^2＋ occur at 227, 241, 265, 335nm, respectively. Synchronous fluorescence spectra exhibit that microenvironment of Trp residue in PPO is more hydrophobic than that of free Trp in water, and that both fluorescences of Trp and Tyr are quenched by Zn^2＋ , but the microenvironments of both Trp and Tyr residues in PPO undergo no changes with Zn^2＋ addition.