Hanlon S, Wong L, Pack G R
Department of Biochemistry, University of Illinois College of Medicine, Chicago 60612, USA.
Biophys J. 1997 Jan;72(1):291-300. doi: 10.1016/S0006-3495(97)78668-5.
Poisson-Boltzmann calculations by Pack and co-workers suggest the presence of regions of increased hydrogen ion density in the grooves of DNA. As an experimental test of this prediction, we have attached proton-sensitive probes, with variable linker lengths, to random-sequence DNA at G sites in the minor groove. The amino groups of beta-alanine, gamma-aminobutyric acid (GABA), and epsilon-aminocaproic acid have been coupled at pH 5, via a formaldehyde link, to the exocyclic amino group of guanine, utilizing a reaction that has been extensively investigated by Hanlon and co-workers. The resulting adducts at pH 5 retained duplex B form but exhibited typical circular dichroism (CD) changes previously shown to be correlated with the presence of a net positive charge in the minor groove. Increases in the solvent pH reversed the CD spectral changes in a manner suggesting deprotonation of the carboxylic acid group of the adduct. These data were used to calculate an apparent pK(a) for the COOH. The pK(a) was increased by 2.4 units for beta-alanine, by 1.7 units for GABA, and by 1.5 units for epsilon-amino caproic acid, relative to their values in the free amino acid. This agrees well with Poisson-Boltzmann calculations and the energy minimization of the structures of the adducts that place the carboxyl groups in acidic domains whose hydrogen ion density is approximately 2 orders of magnitude greater than that of bulk solvent.
帕克及其同事进行的泊松-玻尔兹曼计算表明，在DNA的凹槽中存在氢离子密度增加的区域。作为对这一预测的实验验证，我们将具有可变连接长度的质子敏感探针连接到小沟中G位点的随机序列DNA上。在pH值为5的条件下，利用汉隆及其同事广泛研究过的一种反应，通过甲醛连接将β-丙氨酸、γ-氨基丁酸（GABA）和ε-氨基己酸的氨基与鸟嘌呤的环外氨基偶联。在pH值为5时得到的加合物保持双链B型，但呈现出典型的圆二色性（CD）变化，此前已表明这种变化与小沟中净正电荷的存在相关。溶剂pH值的升高以一种表明加合物羧酸基团去质子化的方式逆转了CD光谱变化。这些数据用于计算COOH的表观pK(a)。相对于它们在游离氨基酸中的值，β-丙氨酸的pK(a)增加了2.4个单位，GABA增加了1.7个单位，ε-氨基己酸增加了1.5个单位。这与泊松-玻尔兹曼计算以及加合物结构的能量最小化结果非常吻合，加合物结构将羧基置于氢离子密度比本体溶剂大约高2个数量级的酸性区域。
