Zhou Hong-Wei, Xu Yan, Zhou Hai-Meng
Department of Biological Science and Biotechnology, Tsinghua University, Beijing, PR China.
Biochem Cell Biol. 2002;80(2):205-13. doi: 10.1139/o02-003.
The effect of trifluoroethanol (TFE) on horseradish peroxidase (HRP) was determined using activity assay and spectral analysis including optical absorption, circular dichroism (CD), and intrinsic fluorescence. The enzyme activity increased nearly twofold after incubation with 5-25% (v/v) concentrations of TFE. At these TFE concentrations, the tertiary structure of the protein changed little, while small changes occurred at the active site. Further increases in the TFE concentration (25-40%) decreased the enzyme activity until at 40% TFE the enzyme was completely inactivated. The alpha-helix content of the protein increased at high TFE concentrations, while near-UV CD, Soret CD, and intrinsic fluorescence indicated that the tertiary structure was destroyed. Polyacrylamide gel electrophoresis results indicated that the surface charge of the enzyme was changed at TFE concentrations greater than 20%, and increasing concentrations of TFE reduced the enzyme molecular compactness. A scheme for the unfolding of HRP in TFE was suggested based on these results. The kinetics of absorption change at 403 nm in 40% TFE followed a two-phase course. Finally, HRP incubated with TFE was more sensitive to urea denaturation, which suggested that the main effect of TFE on HRP was the disruption of hydrophobic interactions.
使用活性测定和光谱分析（包括光吸收、圆二色性（CD）和内源荧光）来确定三氟乙醇（TFE）对辣根过氧化物酶（HRP）的影响。用5-25%（v/v）浓度的TFE孵育后，酶活性增加了近两倍。在这些TFE浓度下，蛋白质的三级结构变化不大，而活性位点发生了小的变化。TFE浓度进一步增加（25-40%）会降低酶活性，直到在40% TFE时酶完全失活。在高TFE浓度下，蛋白质的α-螺旋含量增加，而近紫外CD、Soret CD和内源荧光表明三级结构被破坏。聚丙烯酰胺凝胶电泳结果表明，在TFE浓度大于20%时，酶的表面电荷发生了变化，并且TFE浓度增加会降低酶分子的紧密性。基于这些结果，提出了HRP在TFE中展开的示意图。在40% TFE中403 nm处吸收变化的动力学遵循两相过程。最后，与TFE孵育的HRP对尿素变性更敏感，这表明TFE对HRP的主要作用是破坏疏水相互作用。
