Lin M C, Macgregor R B
Faculty of Pharmacy, University of Toronto, Ontario, Canada.
Biochemistry. 1996 Sep 10;35(36):11846-51. doi: 10.1021/bi9613279.
The role of hydration in the kinetics of a DNA helix-coil equilibrium is investigated by studying the effect of hydrostatic pressure on the rate constants describing the reaction. The kinetics were measured using the thermal hysteresis between the denaturation and renaturation curves of the triplex-forming oligonucleotides: 5'd[AAA-GGAGGAGAAGAAGAAAAAA] (sequence of purine strand) and 5'd[TTTCCTCCTCTTCTTCTTTTTT] (third strand). The kinetics at atmosphere pressure for this system have been recently reported [Rougée et al. (1992) Biochemistry 31, 9269-9278]. At all pressures the data are consistent with a single-step bimolecular reaction under the conditions of our experiments (100 mM NaCl, 10 mM cacodylate, pH 6.5). The rate of formation of the triplex from the duplex + single strand is accelerated by pressure. At the midpoint of the helix-coil transition (32.5 degrees C), the activation volume for helix formation, V1, equals -11.8 (+/- 2.4) cm3 mol-1 at atmospheric pressure. At the same temperature, the activation volume for helix dissociation, V-1, equals +39.9 (+/- 5.0) cm3 mol-1; that is, the rate of strand separation is slowed by pressure. These findings emphasize the importance of solvent interactions in the stabilization and formation of DNA helices. It is proposed that the activation volume of the forward reaction may arise from the volume change due to charging the cytosine residues and the formation of base-stacking interactions in the third strand. The positive activation volume of strand separation may be a consequence of poor solvent packing of the DNA duplex major groove during dissociation of the third strand.
通过研究静水压力对描述反应的速率常数的影响，研究了水合作用在DNA螺旋-线圈平衡动力学中的作用。使用三链体形成寡核苷酸的变性曲线和复性曲线之间的热滞来测量动力学：5'd[AAA-GGAGGAGAAGAAGAAAAAA]（嘌呤链序列）和5'd[TTTCCTCCTCTTCTTCTTTTTT]（第三条链）。该系统在大气压下的动力学最近已有报道[鲁热等人（1992年），《生物化学》31卷，9269 - 9278页]。在所有压力下，数据都与我们实验条件（100 mM氯化钠，10 mM二甲胂酸，pH 6.5）下的单步双分子反应一致。从双链体+单链形成三链体的速率因压力而加快。在螺旋-线圈转变的中点（32.5℃），大气压下螺旋形成的活化体积V1等于-11.8（±2.4）cm³ mol⁻¹。在相同温度下，螺旋解离的活化体积V-1等于+39.9（±5.0）cm³ mol⁻¹；也就是说，链分离的速率因压力而减慢。这些发现强调了溶剂相互作用在DNA螺旋稳定和形成中的重要性。有人提出，正向反应的活化体积可能源于胞嘧啶残基带电和第三条链中碱基堆积相互作用形成所导致的体积变化。链分离的正活化体积可能是第三条链解离过程中DNA双链体大沟溶剂堆积不良的结果。
