Gilbert P L, Graves D E, Britt M, Chaires J B
Department of Chemistry, University of Mississippi, University 38677.
Biochemistry. 1991 Nov 12;30(45):10931-7. doi: 10.1021/bi00109a018.
The photoaffinity analogue ethidium monoazide was used to prepare samples of poly(dGdC).poly(dGdC) containing covalently attached ethidium. The effects of both noncovalently and covalently bound ethidium on the kinetics of the NaCl-induced B to Z transition in poly(dGdC).poly(dGdC) was examined using absorbance and fluorescence spectroscopy to monitor the reaction. Covalently and noncovalently attached ethidium were equal in the extent to which they reduce the rate of the B to Z transition. By using fluorescence to selectively monitor the fate of noncovalently bound ethidium over the course of the transition, we found that ethidium completely dissociates as the reaction proceeds, but at a rate that lags behind the conversion of the polymer to the Z form. These experiments provide evidence for the redistribution of noncovalently bound ethidium over the course of the B to Z transition, leading to the development of biphasic reaction kinetics. The observed kinetics suggest that the primary effect of both covalently and noncovalently bound ethidium is on the nucleation step of the B to Z transition. The reduction in the rate of the B to Z transition by noncovalently or covalently bound ethidium may be quantitatively explained as resulting from the reduced probability of finding a drug-free length of helix long enough for nucleation to occur. As necessary ancillary experiments, the defined length deoxyoligonucleotides (dGdC)4, (dGdC)5, and (dGdC)6 were synthesized and used in kinetic experiments designed to determine the nucleation length of the B to Z transition, which was found to be 6 bp. The activation energy of the B to Z transition was demonstrated to be independent of the amount of covalently bound ethidium and was found to be 21.2 +/- 1.1 kcal mol-1. Covalent attachment of ethidium was observed to increase the rate of the reverse Z to B transition, presumably by locking regions of the polymer into a right-handed conformation and thereby providing nucleation sites from which the Z to B conversion may propagate.
光亲和类似物单叠氮溴化乙锭被用于制备含有共价连接溴化乙锭的聚（dGdC）·聚（dGdC）样品。使用吸光度和荧光光谱监测反应，研究了非共价和共价结合的溴化乙锭对聚（dGdC）·聚（dGdC）中NaCl诱导的B型到Z型转变动力学的影响。共价和非共价连接的溴化乙锭在降低B型到Z型转变速率的程度上是相同的。通过在转变过程中使用荧光选择性地监测非共价结合的溴化乙锭的命运，我们发现随着反应进行，溴化乙锭完全解离，但解离速率落后于聚合物向Z型的转化。这些实验为非共价结合的溴化乙锭在B型到Z型转变过程中的重新分布提供了证据，导致了双相反应动力学的发展。观察到的动力学表明，共价和非共价结合的溴化乙锭的主要作用是在B型到Z型转变的成核步骤上。非共价或共价结合的溴化乙锭导致B型到Z型转变速率降低，可以定量解释为由于找到足够长的无药物螺旋长度以发生成核的概率降低。作为必要的辅助实验，合成了确定长度的脱氧寡核苷酸（dGdC）4、（dGdC）5和（dGdC）6，并用于动力学实验，以确定B型到Z型转变的成核长度，发现为6个碱基对。B型到Z型转变的活化能被证明与共价结合的溴化乙锭的量无关，为21.2±1.1千卡/摩尔。观察到溴化乙锭的共价连接增加了反向Z型到B型转变的速率，推测是通过将聚合物区域锁定为右手构象，从而提供Z型到B型转化可以传播的成核位点。
