The denaturation-renaturation thermal hysteresis was used to investigate the kinetics of the helix-coil equilibrium of four 22-base pair homopurine-homopyrimidine duplex oligonucleotides with fractional G x C base pair content (f(G x C)) between 0.14 and 0.5. In 20 mM NaCl and 20 mM Tris-HCl at pH 7.0 and at hydrostatic pressures up to 200 MPa, a two-state bimolecular reaction mechanism adequately described the observed kinetics. At 1 MPa and 47 degrees C, the rate constant for helix formation, k1, increased by a factor of 210, and the reverse rate constant, k(-1), decreased by a factor of 420 upon increasing f(G x C) from 0.14 to 0.5. The activation energies for formation of the duplexes were negative and relatively insensitive to f(G x C). The pressure-induced change in the rate constants is related to the activation volume of the reaction step. Pressure causes k1 to become larger, and the magnitude of the change in k1 with pressure increases the lower the f(G x C) value. Thus, when f(G x C) = 0.14, the activation volume for forward reaction, delta V++(1), equals -20 mL/mol, while when f(G x C) = 0.5, delta V++(1) = -6.7 mL/mol. The rate constant for strand separation, k(-1), decreases at high pressure. The activation volume for this step, delta V++(1), varies from 17 to 1.6 mL/mol when f(G x C) = 0.14 and 0.5, respectively. The delta V for helix formation calculated from the activation parameters changed from -23 mL/mol when f(G x C) = 0.14 to -5.8 mL/mol when f(G x C) = 0.5. From extrapolation, it is estimated that the molar volume change for formation of G x C base pairs in homopurine-homopyrimidine sequences is approximately 0 mL/mol. Parameters calculated from kinetics of other two duplex molecules, when f(G x C) = 0.23 and 0.32, lie between these extremes.

译文

变性-复性热滞后用于研究0.14和0.5之间具有分数G x C碱基对含量 (f(G x C)) 的四种22碱基对高嘌呤-高嘧啶双链体寡核苷酸的螺旋-螺旋平衡动力学。在pH 7.0和高达200 MPa的静水压力下,在20 mM NaCl和20 mM Tris-HCl中,两态双分子反应机理充分描述了观察到的动力学。在1 mpa和47 ℃ 下,当f(G x C) 从0.14增加到0.5时,螺旋形成的速率常数k1增加了210倍,反向速率常数k(-1) 降低了420倍。形成双链体的活化能为负,并且对f(G x C) 相对不敏感。压力引起的速率常数变化与反应步骤的活化体积有关。压力导致k1变大,并且k1随压力变化的幅度增加,f(G x C) 值越低。因此,当f(G x C) = 0.14时,正向反应的活化体积 δ V ++(1) 等于-20 mL/mol,而当f(G x C) = 0.5时,δ V ++(1) = -6.7 mL/mol。在高压下,链分离的速率常数k(-1) 降低。当f(G x C) = 0.14和0.5时,该步骤的活化体积 Δ V ++(1) 分别从17至1.6 mL/mol变化。由活化参数计算的螺旋形成的 δ V从f (gxc) = 0.14时的-23 mL/mol变化到f (gxc) = 0.5时的-5.8 mL/mol。根据外推法,估计在均嘌呤-高嘧啶序列中形成G x C碱基对的摩尔体积变化约为0毫升/mol。当f(G x C) = 0.23和0.32时,根据其他两个双链体分子的动力学计算出的参数介于这些极端之间。

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