钴离子对烟草蚀纹病毒蛋白酶结构的影响

Study on the impacts of Co^(2+) on the tobacco etch virus protease

烟草蚀纹病毒蛋白酶(Tobacco Etch Virus protease,TEVp)是一种高特异性、高保守的重要工程蛋白,其结构稳定性尤为重要;钴离子(Co^(2+))具有一定的生物毒性.为了解Co^(2+)对TEVp结构的影响,运用内源荧光方法和同步荧光方法探讨Co^(2+)对TEVp结构的调控机理.光谱显示Co^(2+)对TEVp内源荧光有明显的淬灭作用,导致TEVp分子中色氨酸和酪氨酸残基的疏水性增加.在300 K和311 K温度条件下,淬灭常数K_(sv)分别为4.161×10~2L/mol和2.129×10~2L/mol,结合平衡常数K_A分别为6.625×10~3L/mol和5.132×10~3L/mol,且动态荧光淬灭速率常数Kq远大于扩散碰撞淬灭速率常数的最大值2.0×10^(10)L mol^(-1)s^(-1),表明其淬灭机制属于静态淬灭.吉布斯自由能ΔG<0,焓值ΔH<0且熵值ΔS>0,表明Co^(2+)与TEVp的相互作用能够自发进行,且它们之间的主要作用力类型为静电作用力.本研究分析Co^(2+)对TEVp多种荧光光谱性质的影响和Co^(2+)对TEVp结构影响的作用机制,可为TEVp在生物学和生物工程等领域的有效利用奠定基础.

Tobacco etch virus protease（TEVp） is an important enzyme with unique and highly-conserved enzymatic activity. Co（2＋） has special biological toxicity. In order to understand the influence of Co（2＋） on the structure of TEVp, we determined the intrinsic fluorescence emission and synchronous fluorescence spectroscopies reflecting the regulation of TEVp structure by Co2. The fluorescence emission and the synchronous fluorescence spectra suggested that Co（2＋） quenched the fluorescence of TEVp significantly and led to the increase in the hydrophobicity of tryptophan and tyrosine residues. Under the conditions of 300 K and 311 K, the quenching constant K_（sv） was found to be 4.161×10^2 L/mol and 2.129×10^2 L/mol, respectively; the binding constant K_A was 6.625×10^3 L/mol and 5.132×10^3 L/mol, respectively. Moreover, the dynamic fluorescence quenching rate constant K q was much greater than the maximum value of the diffusion collision quenching rate constant, 2.0×10（10） L mol（-1） s（-1）, suggesting the quenching mechanism was static quenching. The Gibbs free energy ΔG 0, the enthalpy value ΔH 0 and the entropy value ΔS 0, indicated the spontaneous interaction between Co（2＋） and TEVp, and that electrostatic force played a major role when Co（2＋） binds to TEVp. Our studies analyzed the influences of Co（2＋） on various fluorescence spectra of TEVp and revealed the mechanism of the impacts of Co（2＋） on the structure of TEVp, thus laying the ground for the efficient utilization of TEVp in the fields of biology and biological engineering.