College of Physics and Chemistry, Henan Polytechnic University, Jiaozuo 454003, China.
Appl Spectrosc. 2013 Jun;67(6):648-55. doi: 10.1366/12-06793.
The molecular mechanism of the interaction between pepsin and two typical ionic liquids (ILs), 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) and 1-octyl-3-methylimidazolium chloride ([C8mim]Cl), was investigated with fluorescence spectroscopy, ultraviolet absorption, and circular dichroism spectroscopy at a pH value of 1.6. The results suggest that ILs could quench the intrinsic fluorescence of pepsin, probably via a dynamic quenching mechanism. The fluorescence quenching constants were determined by employing the classic Stern-Volmer equation. The constant values are very small, indicating that only a very weak interaction between ILs and pepsin exists. The Gibbs free-energy change, enthalpy change (ΔH), and entropy change (ΔS) during the interaction of pepsin and ILs were estimated. Positive values of ΔH and ΔS indicate that the interaction between ILs and pepsin is mainly driven by hydrophobic interaction. Synchronous and three-dimensional fluorescence spectra demonstrate that the addition of ILs (0-0.20 mol L(-1) for each IL) does not bring apparent changes to the microenvironments of tyrosine and tryptophan residues. Activity experiments show that the activity of pepsin is concentration dependent; higher concentrations of ILs (>0.22 mol L(-1) for [C8mim]Cl and >0.30 mol L(-1) for [C4mim]Cl) cause the remarkable reduction of enzyme activity. The presence of ILs also does not improve the thermal stability of pepsin.
在 pH 值为 1.6 时，用荧光光谱法、紫外吸收光谱法和圆二色性光谱法研究了胃蛋白酶与两种典型的离子液体（ILs），1-丁基-3-甲基咪唑氯([C4mim]Cl)和 1-辛基-3-甲基咪唑氯([C8mim]Cl)之间相互作用的分子机制。结果表明，ILs 可以猝灭胃蛋白酶的固有荧光，可能通过动态猝灭机制。通过经典的 Stern-Volmer 方程确定了荧光猝灭常数。常数值很小，表明 ILs 和胃蛋白酶之间只有非常弱的相互作用。还估算了胃蛋白酶与 ILs 相互作用过程中的吉布斯自由能变化（ΔG）、焓变（ΔH）和熵变（ΔS）。ΔH 和 ΔS 的正值表明 ILs 与胃蛋白酶之间的相互作用主要是由疏水相互作用驱动的。同步和三维荧光光谱表明，添加 ILs（每种 IL 为 0-0.20 mol·L-1）不会使酪氨酸和色氨酸残基的微环境发生明显变化。活性实验表明，胃蛋白酶的活性与浓度有关；较高浓度的 ILs（[C8mim]Cl 大于 0.22 mol·L-1，[C4mim]Cl 大于 0.30 mol·L-1）会导致酶活性显著降低。ILs 的存在也不能提高胃蛋白酶的热稳定性。
