Hydrogen--deuterium exchange analysis of ligand--macromolecule interactions: ethidium--deoxyribonucleic acid system.
Hydrogen--deuterium exchange analysis of ligand--macromolecule interactions: ethidium--deoxyribonucleic acid system.
复制标题
配体-大分子相互作用的氢-氘交换分析：乙锭-脱氧核糖核酸体系。
DOI：
10.1021/bi00566a025
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发表时间：
1980
期刊：
影响因子：
2.9
通讯作者：
Kallenbach,NR
中科院分区：
文献类型：
--
作者：
Mandal,C;Englander,SW;Kallenbach,NR
C.曼达尔，S。W. Englander和N. R. Kallenbach* 摘要：采用停流模式下的荧光光谱法研究了DNA与嵌入染料溴化乙锭的相互作用。该方法依赖于这样的事实，即乙锭H交换大大减缓与DNA的络合和HD交换动力学的发色团可以通过其光谱吸光度的伴随变化进行监测。表征了乙锭的HD交换相关光谱特征，研究了游离染料和染料-DNA复合物的催化交换行为。通过这种测量，可以获得相互作用的速率和平衡常数，还可能获得一些立体化学信息。在更常规的混合实验中检查所获得的常数。在20 ℃高盐条件下，平衡结合常数约为5 × 104 M ~（-1），开和关速率常数分别为1.6 × 104 M ~（-1）和30 ~ 4S ~（-1）。结果独立地证实了每2-2.5个碱基对结合一个染料分子。该方法应适用于一系列结合相互作用。在其他优点中，这种方法可以允许在远高于特征值的反应物浓度下研究紧密结合的相互作用。本文报道了杀锥虫染料溴化乙锭与DNA的嵌入结合作用。在停流实验中，我们通过在540 nm附近的可见光谱中的吸光度变化监测了乙锭中氨基质子的氢-氘交换（结构如图2所示）。结果产生的平衡和动力学常数络合的乙锭的DNA，这些常数进行了验证，在更传统的停流mixing experiments.Information配体和大分子之间的复合物最经常来自结合的研究，依赖于分区自由ligandfrom绑定配体或建立一个光谱特征的复杂的是不同于自由形式。本工作表明，发色配体可交换氢的停流动力学测量可用于确定络合的平衡和动力学参数，并可间接提供有关复合物中结合相互作用的立体化学的信息。一个类似的AP-
C. Mandal, S. W. Englander, and N. R. Kallenbach* abstract: The interaction between DNA and the interca-lating dye, ethidium bromide, was studied by use of a novel approach in which hydrogen-deuterium (HD) exchange between ethidium amino groups and solvent was measured spectrophotometrically in a stopped-flow mode. The method depends on the fact that ethidiumH exchange is greatly slowed on complexation with DNA and that HD exchange kinetics of the chromophore can be monitored via an accompanying change in its spectral absorbance. The HD exchange dependent spectral character of ethidium was characterized, and the catalyzed exchange behavior of the free dye and the dye-DNA complex was studied. From such measurements, one can obtain rate and equilibrium constants for the inter-action and possibly also some stereochemical information. The constants obtained were checked in more conventional mixing experiments. At 20 C in high salt, the equilibrium binding constant is~ 5 X 104 M'\and on and off rate constants are 1.6 X 10s M'1 s'1 and 30s'1, respectively. The results independently confirm that one dye molecule is bound for each 2-2.5 base pairs. The method should be applicable to a range of binding interactions. Among other advantages, this approach can allow tight binding interactions to be studied at concentrations of the reactants far above thecharacteristic value. is paper reports on studies of the intercalative binding of the trypanocidal dye ethidium bromide to DNA. In stop-ped-flow experiments, we monitored the hydrogen-deuterium exchange of amino protons in ethidium (structure shown in Figure 2) by means of an absorbance change in the visible spectrum near 540 nm. The results yield equilibrium and kinetic constants for complexation of ethidium by DNA, and these constants were validated in more conventional stopped-flow mixing experiments.Information about complexes between ligands and macro-molecules has been most often derived from binding studies that rely either on partitioning free ligandfrom bound ligand or on establishing a spectroscopic feature of the complex that is distinct from the free forms. The present work shows that the stopped-flow kinetic measurement of exchangeable hydrogens of a chromophoric ligand can be used to determine both equilibrium and kinetic parameters of complexation and may indirectly provide information about the stereochemistry of binding interactions in the complex. An analogous ap-