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首页> 外文期刊>Spectrochimica acta, Part A. Molecular and biomolecular spectroscopy >Two-dimensional fluorescence correlation spectroscopy IV: Resolution of fluorescence of tryptophan residues in alcohol dehydrogenase and lysozyme
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Two-dimensional fluorescence correlation spectroscopy IV: Resolution of fluorescence of tryptophan residues in alcohol dehydrogenase and lysozyme

机译：二维荧光相关光谱法IV：酒精脱氢酶和溶菌酶中色氨酸残基的荧光分辨

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摘要

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve the fluorescence spectra of two tryptophan (Trp) residues in alcohol dehydrogenase and lysozyme. In each protein, one Trp residue is buried in a hydrophobic domain of the protein matrix and the other Trp residue is located at a hydrophilic domain close to the protein-water interface. Fluorescence quenching by iodide ion, a hydrophilic quencher, was employed as a perturbation to induce the intensity change in the spectra. The Trp residue which is located at the hydrophilic domain is effectively quenched by the quencher, while the Trp residue located at the hydrophobic domain is protected from the quenching. Therefore, the fluorescence of these two Trp residues have a different sensitivity to the quenching, showing a different response to the concentration of the quencher. Fluorescence spectra of the two Trp residues in alcohol dehydrogenase, which are heavily overlapped in conventional one-dimensional spectra, have been successfully resolved by the 2D correlation technique. From the asynchronous correlation map, it was revealed that the quenching of Trp located at the hydrophobic part was brought about after that of Trp located at the hydrophilic part. In contrast, the fluorescence spectra of the two Trp residues could not be resolved after the alcohol dehydrogenase was denatured with guanidine hydrochloride. These results are consistent with the well-known structure of alcohol dehydrogenase. Furthermore, it was elucidated that the present 2D analysis is not interfered by Raman bands of the solvent, which sometimes bring difficulty into the conventional fluorescence analysis. Fluorescence spectra of the Trp residues in lysozyme could not be resolved by the 2D correlation technique. The differences between the two proteins are attributed to the fact that the Trp residue in the hydrophobic site of lysozyme is not sufficiently protected from the quenching. (c) 2005 Elsevier B.V. All rights reserved.
机译：广义二维（2D）荧光相关光谱已用于解析酒精脱氢酶和溶菌酶中两个色氨酸（Trp）残基的荧光光谱。在每种蛋白质中，一个Trp残基掩埋在蛋白质基质的疏水域中，另一个Trp残基位于靠近蛋白质-水界面的亲水域中。通过碘离子（一种亲水性猝灭剂）进行的荧光猝灭被用作扰动，以引起光谱强度的变化。位于亲水结构域的Trp残基可被淬灭剂有效淬灭，而位于疏水结构域的Trp残基可免受淬灭作用。因此，这两个Trp残基的荧光对猝灭的敏感性不同，显示出对猝灭剂浓度的不同响应。醇脱氢酶中两个Trp残基的荧光光谱在常规的一维光谱中非常重叠，已通过2D相关技术成功解析。从异步相关图可知，位于疏水部分的Trp的淬灭是位于位于亲水部分的Trp的淬灭之后进行的。相反，醇脱氢酶用盐酸胍变性后，两个Trp残基的荧光光谱无法分辨。这些结果与醇脱氢酶的众所周知的结构一致。此外，已经阐明了本发明的2D分析不受溶剂的拉曼带的干扰，这有时给常规的荧光分析带来困难。二维相关技术无法解析溶菌酶中Trp残基的荧光光谱。两种蛋白质之间的差异归因于以下事实：溶菌酶的疏水位点中的Trp残基没有得到充分的保护以免受淬灭。 （c）2005 Elsevier B.V.保留所有权利。

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