Hungerford Graham, Rei Ana, Ferreira M Isabel C
Departamento de Física, Universidade do Minho, Braga, Portugal.
FEBS J. 2005 Dec;272(23):6161-9. doi: 10.1111/j.1742-4658.2005.05019.x.
The interaction of an extrinsic probe (Nile red) with an enzyme (horseradish peroxidase) in solution was investigated using fluorescence techniques. Nile red fluorescence is very environmentally sensitive and the presence of domains of differing polarity within the enzyme was ascertained by the decomposition of the Nile red emission spectrum. Further evidence for the position of the probe inside the enzyme was obtained from a molecular modeling study. A decrease in the emission intensity of the dye during incubation with horseradish peroxidase was explained by the occurrence of resonance energy transfer between the Nile red and the heme group in the enzyme. This was supported by a calculation of the critical transfer distance and a comparison of the fluorescence intensity of the dye in both the holo- and apo-enzyme. These data were then applied to the study of the effect of temperature on the structure of the enzyme, where changes in conformation were elucidated.
利用荧光技术研究了溶液中外源探针（尼罗红）与酶（辣根过氧化物酶）之间的相互作用。尼罗红荧光对环境非常敏感，通过尼罗红发射光谱的分解确定了酶内不同极性区域的存在。分子建模研究进一步获得了探针在酶内部位置的证据。与辣根过氧化物酶孵育期间染料发射强度的降低是由尼罗红与酶中血红素基团之间发生的共振能量转移所解释的。这通过临界转移距离的计算以及染料在全酶和脱辅基酶中的荧光强度比较得到了支持。然后将这些数据应用于研究温度对酶结构的影响，阐明了构象变化。
