Interaction of ethidium bromide with whole and selectively deproteinized deoxynucleoproteins from calf thymus.
Interaction of ethidium bromide with whole and selectively deproteinized deoxynucleoproteins from calf thymus.
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溴化乙锭与小牛胸腺中完整且选择性脱蛋白的脱氧核蛋白的相互作用。
DOI：
10.1016/0022-2836(72)90444-5
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发表时间：
1972
影响因子：
5.6
通讯作者：
Evangelos N. Moudrianakis
中科院分区：
文献类型：
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作者：
Lynne M. Angerer;Evangelos N. Moudrianakis
我们通过分光光度和荧光技术研究了荧光染料溴化乙锭与 DNA、脱氧核糖核蛋白 (DNP) 以及已去除特定种类蛋白质的 DNP 的结合。根据 Scatchard (1949) 理论分析平衡结合实验的数据，并比较完整和部分脱氧核糖核蛋白的可用结合位点的数量和关联常数的大小。当蛋白质从 DNP 中去除时，观察到缔合常数增加以及可用结合位点数量增加。此外，当从 DNP 中去除的蛋白质是通过 0.6m-NaCl 提取的蛋白质（主要包括组蛋白 f1）时，这两个结合参数的变化幅度最大。根据溴化乙锭与天然 DNP 的结合计算出的缔合常数与报告的染料与人工重建的 DNA-f1 复合物的结合的缔合常数显着不同，因此可能反映了天然和人工核蛋白之间的重要结构差异。通过检查与各种核蛋白样品结合的染料的荧光发射光谱和量子产率，进一步研究了结合位点环境的性质。尚未观察到这些参数中的任何一个存在差异。此外，还通过荧光测定法检查了离子强度对溴化乙锭结合的影响，并根据可能的结合机制进行了讨论。根据其沿 DNA 组织的各种模型，讨论了染色体蛋白限制 DNP 中 DNA 染料结合能力的方式。此外，与部分 DNP 和 DNA 相比，整个 DNP 对溴化乙锭的亲和力降低是由特定种类或临界量的蛋白质造成的。 DNP 的这种改变的染料结合能力可能是对可用结合位点的不完全限制的表现，或者是新的、结构上不同的位点的出现，这些位点不会改变结合的染料分子的吸收或荧光特性。
We have investigated by means of both spectrophotometric and fluorometric techniques the binding of the fluorescent dye, ethidium bromide, to DNA, to deoxyribonucleoproteins (DNP) and to DNP's from which specific kinds of proteins have been removed. The data from equilibrium binding experiments are analyzed according to Scatchard (1949) theory, and the number of available binding sites and the magnitude of the association constants are compared for whole and partial deoxyribonucleoproteins. As protein is removed from DNP, an increase in the association constant as well as an increase in the number of avaiable binding sites is observed. Furthermore, the magnitude of the changes of both of these binding parameters is largest when the proteins removed from DNP are those extracted by 0.6m-NaCl which primarily include histone f1. The association constant calculated from the binding of ethidium bromide to native DNP differs significantly from that reported for the binding of the dye to artificially reconstituted DNA-f1 complexes and therefore may reflect important structural differences between native and artificial nucleoproteins.The nature of the environment of the binding sites is further studied by examining the fluorescence emission spectrum and the quantum yield of dye bound to the various nucleoprotein samples. No differences in either of these parameters have been observed. In addition, the effect of ionic strength on ethidium bromide binding is examined fluorometrically and discussed in terms of the probable binding mechanism.The manner by which chromosomal proteins limit the dye-binding capacity of DNA in DNP is discussed with respect to various models of their organization along the DNA. Furthermore, it is suggested that either a specific kind or critical amount of protein is responsible for the reduced affinity for ethidium bromide of whole DNP compared to that of partial DNP's and DNA. This altered dye binding capacity of DNP may be a manifestation of an incomplete restriction of available binding sites or of the appearance of new, structurally distinct sites which do not change the absorption or fluorescence properties of bound dye molecules.