Cross D G
Biochemistry. 1975 Jan 28;14(2):357-62. doi: 10.1021/bi00673a023.
Time-dependent changes in the ultraviolet absorbance of the adenine chromophore are observed in the stopped-flow spectrophotometer when adenosine and its analogs are rapidly transferred from protium oxide to deuterium oxide. These absorbance changes are shown to result from hydrogen exchange in the exocyclic amino groups of the purine ribonucleosides by using derivatives of adenosine in which methyl groups replace exchangeable hydrogens and by showing that the general characteristics of hydrogen exchange in adenosine analogs agree with those found here. A study of the dependence of hydrogen-exchange rate constants on adenosine, AMP, and phosphate concentration showed there is a second-order dependence on AMP concentration which is primarily due to intermolecular catalysis by the phosphate group of the nucleotide. The deuterium oxide perturbation difference spectrum, obtained at equilibrium, was found to contain two components that result from blue shifts of the adenine chromophore absorbance: (1) a shift cause by the substitution of deuterium for protium in the ring (N1) nitrogen and exocyclic nitrogens, and (2) a shift associated with a change in the polarizability of the medium. Since the theory of solvent perturbation, which is used to measure the relative "exposure" of chromophores in macromolecules, assumes that the spectral shifts observed are solely due to (2) above, the use of deuterium oxide as a measure of chromophore exposure to perturbants the size of water must be reexamined.
当腺苷及其类似物在停流分光光度计中从氧化氕迅速转移至氧化氘时，可观察到腺嘌呤发色团紫外吸光度随时间的变化。通过使用甲基取代可交换氢的腺苷衍生物，并表明腺苷类似物中氢交换的一般特征与本文所发现的一致，这些吸光度变化被证明是由嘌呤核糖核苷环外氨基的氢交换引起的。对氢交换速率常数与腺苷、AMP和磷酸盐浓度的依赖性研究表明，对AMP浓度存在二级依赖性，这主要是由于核苷酸磷酸基团的分子间催化作用。在平衡状态下获得的氧化氘微扰差光谱被发现包含由腺嘌呤发色团吸光度蓝移产生的两个成分：(1) 由于环（N1）氮和环外氮中的氘取代了氕而导致的位移，以及 (2) 与介质极化率变化相关的位移。由于用于测量大分子中发色团相对“暴露程度”的溶剂微扰理论假设观察到的光谱位移仅归因于上述 (2)，因此必须重新审视使用氧化氘作为衡量发色团对水大小的扰动剂暴露程度的方法。
