Effects of various salts and pH on the stability of the nucleosome in chromatin fragments.
Effects of various salts and pH on the stability of the nucleosome in chromatin fragments.
复制标题
各种盐和 pH 对染色质片段中核小体稳定性的影响。
DOI：
10.1021/bi00197a031
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Cole,RD
中科院分区：
文献类型：
--
作者：
Ni,X;Cole,RD
1994年5月27日收到的修订版Mandalpt ®摘要：在广泛的溶液条件下，通过差示扫描量热法观察长染色质片段中核小体的稳定性。染色质的热变性通常被表征为三个主要的转变，尽管该过程显然更复杂。三个主要的转变是（1）核小体的变性，（2）在所得变性核蛋白中DNA的碱基解堆积，和（3）裸DNA的碱基解堆积。在非常低的盐浓度下（例如，2 mM二甲胂酸钠），这三个过程基本上是一致的（接近76 ℃），但在中等盐浓度下（例如，100 mM NaCl），核小体变性首先发生在约69 ℃，然后碱基解堆积发生在85 ℃。随着[NaCl]的增加，所有三个过程都得到解决，观察到裸DNA被熔化的量增加，直到在2000 mM NaCl的量热曲线显示主要是DNA的熔化。随着盐浓度从1增加到600 mM，核小体变性的转变温度从76 ℃降低到63 ℃。增加[NaCl]引起的核小体不稳定性在100 mM以上也是明显的，这是由于核小体变性引起的蛋白质变化的降低。类似地，当[NaCl]增加到100 mM以上时，越来越少的变性核蛋白是明显的，因为越来越多的DNA熔化为裸DNA。脂肪酸盐，戊酸钠和己酸钠，不稳定的核小体，但不变性的核蛋白，导致核小体的崩溃。在系列乙酸酯，丁酸酯，戊酸酯，己酸酯中，很明显，核小体的不稳定性随着疏水性（链长）的增加而增加。庚二酸与己酸具有相同的碳原子数，但多带一个负电荷，它不会像己酸那样使核小体不稳定。在0.5-2 mM范围内的MgCl 2和在0.1- 3 mM范围内的亚精胺基本上稳定核小体。变性核蛋白中DNA的转变温度不受亚精胺的影响，但被MgCl 2降低。亚精胺比MgCl_2更有效地置换变性核蛋白中的DNA.以前，我们报道过（Jin&科尔，1986; Guo &科尔1989 a，B）染色质的凝聚受特定离子和生理范围内pH的强烈影响.这清楚地表明了在比较染色质功能和动力学报告时注意缓冲液组成的重要性。当核小体包含在染色质中时，这些因素也可能对核小体的结构产生实质性影响，但似乎只有适度的关注。这里介绍的工作是探索盐和pH值对核小体染色质大片段稳定性的影响。用于观察稳定性的技术是差示扫描量热法，因为即使在发生沉淀的pH和离子条件的生理范围内，该技术也适用于染色质。
Revised Manuscript Received May 27, 1994® abstract: The stability of nucleosomes in long chromatin fragments was observed by differential scanning calorimetry over a wide range of solution conditions. The thermal denaturation of chromatin was characterized in general as three major transitions, although the process clearly is more complex. The three major transitions were (1) denaturation of the nucleosome,(2) base unstacking of DNA in the resulting denatured nucleoprotein, and (3) base unstacking of naked DNA. In very low salt concentrations (eg, 2 mM sodium cacodylate), these three processes were essentially coincident (near 76 0 C), but in medium salt concentrations (eg, 100 mM NaCl) the nucleosome denaturation occurred first at about 69 C and then base unstacking occurred at 85 C. As [NaCl] was increased, all three processes were resolved with the observation of increasing amounts of naked DNA being melted, until at 2000 mM NaCl the calorimetric profile showed mainly the melting of DNA. The transition temperature for nucleosome denaturation decreased from 76 to 63 C as the salt concentration increased from 1 to 600 mM. Destabilization of the nucleosome by increasing [NaCl] was also evident above 100 mM as a decrease in enthalpic change attributable to nucleosome denaturation. Similarly, as [NaCl] was increased above 100 mM, less and less denatured nucleoprotein was evident as more and more of the DNA melted as naked DNA. The fatty acid salts, sodium valerate and sodium caproate, destabilized the nucleosome but not the denatured nucleoprotein that resulted from the collapse of the nucleosome. In the series acetate, butyrate, valerate, caproate, it was clear that destabilization of the nucleosome increased as hydrophobicity (chain length) increased. Pimelate, with the same number of carbon atoms as caproate butwith an extra negative charge, did not destabilize the nucleosome as caproate did. The nucleosome was substantially stabilized by MgCl2 within the range 0.5-2 mM and by spermidine in the range 0.1-3mM. The transition temperature for DNA in the denatured nucleoprotein was unaffected by spermidine, but was lowered by MgCl2. Spermidine was more effective than MgCl2 at displacing DNA from the denatured nucleoprotein.Previously, we reported (Jin& Cole, 1986; Guo & Cole 1989a, b) that the condensation of chromatin was strongly affected by particular ions and by pH inthe physiological range. This made clear the importance of paying attention to buffer composition when comparing reports of chromatin function and dynamics. Such factors might also have substantial effects on the structure of the nucleosome when it is contained within chromatin, but only modest attention seems to have been given to that possibility. The work to be presented here was an exploration of salt and pH effects on the stability of the nucleosomein large fragments of chromatin. The technique used to observe stability was differential scanning calorimetry because it is applicable to chromatin even in the physiological range of pH and ionic conditions where precipitation occurs.