Hadfield A, Hajdu J
Laboratory of Molecular Biophysics, Oxford University, U.K.
J Mol Biol. 1994 Mar 4;236(4):995-1000. doi: 10.1016/0022-2836(94)90006-x.
In time-resolved diffraction studies, reaction initiation should ideally be both uniform throughout the body of the crystal and rapid with respect to the reaction under study. Caged compounds have been used in a number of experiments to provide photochemical initiation of catalytic reactions in enzyme crystals. No in situ measurements have been reported so far on the kinetics of photolysis or on the distribution of photolysis products within crystals. With the aid of a fast single-crystal microspectrophotometer, we performed quantitative studies on the photolysis of a caged compound, 3,5-dinitrophenyl phosphate, in crystals of glycogen phosphorylase b. The results show that for concentrations required in kinetic experiments, the photolytic release of phosphate from 3,5-dinitrophenyl phosphate is restricted to a thin surface layer only. The liberated substrate is then transported by diffusion into the body of the crystal. In effect, the speed of reaction initiation is limited by the rate of diffusion rather than by the rate of the photochemical reaction. The paper discusses general criteria and experimental strategies for the successful use of photoreactive protective groups in time-resolved diffraction experiments.
在时间分辨衍射研究中，理想情况下反应起始应在整个晶体中均匀进行，并且相对于所研究的反应要迅速。笼形化合物已在许多实验中用于在酶晶体中提供催化反应的光化学起始。到目前为止，尚未有关于光解动力学或光解产物在晶体内分布的原位测量报道。借助快速单晶显微分光光度计，我们对糖原磷酸化酶b晶体中的笼形化合物3, ,5 - 二硝基苯磷酸酯的光解进行了定量研究。结果表明，对于动力学实验所需的浓度，3, ,5 - 二硝基苯磷酸酯中磷酸的光解释放仅局限于一个薄的表面层。然后释放的底物通过扩散进入晶体内部。实际上，反应起始的速度受扩散速率限制，而非光化学反应速率。本文讨论了在时间分辨衍射实验中成功使用光反应性保护基团的一般标准和实验策略。
