Yu S S, Li H J, Shih T Y
Biochemistry. 1976 May 18;15(10):2034-41. doi: 10.1021/bi00655a002.
Circular dichroism (CD) was used to investigate the conformations of arginine-rich histones, H3 (III or f3) and H4 (IV or f2a1), and DNA in the complexes prepared by four different methods: (A) NaCl gradient dialysis with urea; (B) NaCl gradient dialysis without urea; (C) direct mixing in 2.5 x 10(-4) M EDTA, pH 8.0; and (D) direct mixing in 0.01 M sodium phosphate, pH 7.0. Using the CD spectrum of native chromatin as a criterion to judge the closeness of a complex to its native state, it was observed that a complex made by direct mixing at low ionic strength (methods C and D) is better than the ones made by NaCl gradient dialysis with or without urea (methods A and B). It is explained as a result of lack of ordered secondary structures in histones due to the presence of urea in method A or due to nonspecific aggregation in NaCl without urea (method B). Compared with all the earlier reports in literature on the CD of histone-DNA complexes, the CD spectra of arginine-rich histone-DNA complexes prepared by methods C and D are closest to that of native chromatin both in shape and in amplitude. These results imply (a) that arginine-rich histones play an important role in maintaining the conformation of chromatin and (b) that the binding of these two histones to DNA prepared by methods C and D are close to that in native chromatin. Noticeable variation in conformation of free and bound histone and histone-bound DNA has also been observed in histone H3 with one or two cysteine residues, and in reduced or oxidized state even when the complexes were prepared and examined in the same condition. CD spectra of arginine-rich histones in 0.01 M phosphates, pH 7.0, indicate the presence of alpha-helix which could be responsible for a favorable binding of the less basic regions of these histones to DNA under this condition as demonstrated by thermal denaturation (Yu, S. .S, Li H. J., and Shih, T. Y. (1976), Bio-chemistry, the preceding paper in this issue). To preserve or generate alpha-helical structures in histones seems to be a critical step in reconstituting good histone-DNA complexes.
利用圆二色性（CD）研究了富含精氨酸的组蛋白H3（III或β3）和H4（IV或f2a1）以及通过四种不同方法制备的复合物中的DNA构象：（A）含尿素的NaCl梯度透析；（B）不含尿素的NaCl梯度透析；（C）在pH 8.0的2.5×10⁻⁴ M EDTA中直接混合；（D）在pH 7.0的0.01 M磷酸钠中直接混合。以天然染色质的CD光谱作为判断复合物与其天然状态接近程度的标准，观察到在低离子强度下直接混合制备的复合物（方法C和D）优于用含或不含尿素的NaCl梯度透析制备的复合物（方法A和B）。这被解释为方法A中由于尿素的存在导致组蛋白中缺乏有序的二级结构，或者方法B中在不含尿素的NaCl中存在非特异性聚集。与文献中所有关于组蛋白-DNA复合物CD的早期报道相比，通过方法C和D制备的富含精氨酸的组蛋白-DNA复合物的CD光谱在形状和幅度上都最接近天然染色质。这些结果表明：（a）富含精氨酸的组蛋白在维持染色质构象中起重要作用；（b）通过方法C和D制备的这两种组蛋白与DNA的结合接近天然染色质中的结合。在含有一个或两个半胱氨酸残基的组蛋白H3中，以及即使在相同条件下制备和检测复合物时处于还原或氧化状态，也观察到游离和结合组蛋白以及组蛋白结合DNA的构象有明显变化。在pH 7.0的0.01 M磷酸盐中富含精氨酸的组蛋白的CD光谱表明存在α-螺旋，如热变性所示（Yu, S. S., Li H. J., and Shih, T. Y. (1976), Bio-chemistry, 本期前文），这可能是这些组蛋白碱性较弱区域在此条件下与DNA良好结合的原因。在组蛋白中保留或生成α-螺旋结构似乎是重构良好的组蛋白-DNA复合物的关键步骤。
