Corin A F, Jovin T M
Biochemistry. 1986 Jul 15;25(14):3995-4007. doi: 10.1021/bi00362a004.
The delayed fluorescence properties of proflavin have been exploited in studies of the excited-state binding kinetics of the dye to poly[d(A-T)] and its brominated analogue poly[d(A-br5U)] at room temperature and pH 7. The two analyzed luminescence decay times of the DNA-dye complex are dependent on the total nucleic acid concentration. This dependence is shown to reflect a temporal coupling of the intrinsic delayed emission decay rates with the dynamic chemical kinetic binding processes in the excited state. Temperature-jump kinetic studies conducted on the brominated polymer and corresponding information on poly[d(A-T)] from a previous study [Ramstein, J., Ehrenberg, M., & Rigler, R. (1980) Biochemistry 19, 3938-3948] provide complementary information about the ground state. In the ground state, the poly[d(A-T)]-proflavin complex has one chemical relaxation time, which reaches a plateau at high DNA concentrations. The brominated DNA-dye complex exhibits two relaxation times: a faster relaxation mode that behaves similarly to that for the unhalogenated DNA and a slower relaxation mode that is apparent at high DNA concentrations. The ground-state kinetic data are analyzed in terms of two alternative models incorporating series and parallel reaction schemes. The former consists of two sequential binding steps--a fast bimolecular process followed by a monomolecular step--while the latter consists of two coupled bimolecular steps. A similar analysis for the excited-state data yields reasonable kinetic constants only for the series model, which, in accordance with previous proposals for acridine intercalators, consists of a fast outside binding step followed by intercalation of the dye. A comparison of the ground- and excited-state kinetic parameters reveals that the external binding process is much stronger and the intercalation is much weaker in the excited state. That the excited-state data are only consistent with the series model suggests that delayed luminescence studies may provide a general tool for distinguishing between the two kinetic mechanisms. In particular, we demonstrate the use of delayed luminescence spectroscopy as a tool for probing dynamic DNA-ligand interactions in solution.
在室温及pH 7条件下，吖啶黄的延迟荧光特性已被用于研究该染料与聚[d(A-T)]及其溴化类似物聚[d(A-br5U)]的激发态结合动力学。DNA-染料复合物的两个分析发光衰减时间取决于总核酸浓度。这种依赖性表明，内在延迟发射衰减率与激发态中的动态化学动力学结合过程存在时间耦合。对溴化聚合物进行的温度跳跃动力学研究以及先前一项研究[Ramstein, J., Ehrenberg, M., & Rigler, R. (1980) Biochemistry 19, 3938 - 3948]中关于聚[d(A-T)]的相应信息，提供了关于基态的补充信息。在基态下，聚[d(A-T)]-吖啶黄复合物有一个化学弛豫时间，在高DNA浓度时达到平稳状态。溴化DNA-染料复合物表现出两个弛豫时间：一个较快的弛豫模式，其行为与未卤化DNA的类似；另一个较慢的弛豫模式，在高DNA浓度时明显。基态动力学数据根据包含串联和平行反应方案的两种替代模型进行分析。前者由两个连续的结合步骤组成——一个快速的双分子过程，随后是一个单分子步骤——而后者由两个耦合的双分子步骤组成。对激发态数据进行类似分析时，仅串联模型得出了合理的动力学常数，根据先前对吖啶嵌入剂的提议，该模型由一个快速的外部结合步骤，随后是染料的嵌入组成。基态和激发态动力学参数的比较表明，在激发态下外部结合过程更强，而嵌入过程更弱。激发态数据仅与串联模型一致，这表明延迟发光研究可能为区分这两种动力学机制提供一种通用工具。特别是，我们展示了延迟发光光谱作为一种工具，用于探测溶液中动态的DNA-配体相互作用。
