烟酰胺腺嘌呤二核苷酸(NADH)是生物体内重要的辅酶分子, 在细胞能量代谢中发挥着关键作用。 金属离子可以影响NADH所参与的酶促反应, 其中铝离子（Al3+）对神经系统具有毒性, 可以引发神经退行性疾病。 因此, Al3+和NADH分子间相互作用的研究有助于了解Al3+对生物体内TCA循环和酶促反应的影响, 具有重要的生物学意义。 本文采用紫外-可见吸收和稳态荧光光谱, 结合时间相关单光子计数技术(TCSPC), 研究了Al3+对水溶液中NADH的本征荧光光谱和分子构象变化的影响。 紫外-可见吸收光谱显示, NADH与Al3+的结合不会改变NADH分子腺嘌呤和烟酰胺两个本征发色团的吸收特性。 为避免NADH分子内两个本征发色团之间的荧光共振能量转移效应的影响, 采用340 nm作为激发波长, 比较了NADH与Al3+作用前后的荧光特性。 实验结果证实, Al3+可以与NADH焦磷酸盐桥上的两个氧原子相结合, 使NADH分子的结构变得相对更加刚性, 从而抑制NADH分子在溶液中的转动等非辐射过程, 导致NADH分子平均荧光寿命增加, 最终引起NADH分子荧光强度随Al3+浓度的增加而线性增强。 进一步, 采用NADH本征荧光寿命振幅比的研究方法表征了NADH分子在溶液中的两种主要构象形式: 腺嘌呤和烟酰胺相互堆积的折叠构象以及腺嘌呤和烟酰胺相互分离的展开构象。 研究发现, Al3+会打破溶液中NADH分子展开构象和折叠构象的平衡状态, 促使辅酶NADH分子的展开构象转变为折叠构象, 最终达到新的动态平衡, 并且当NADH和Al3+以不大于1∶2的浓度比结合时, NADH分子两种构象的振幅比与铝离子浓度的对数间存在线性关系, 在Al3+浓度检测等领域具有良好的应用前景。

Nicotinamide adenine dinucleotide (reduced form) (NADH), as an important coenzyme molecule in enzymes, is ubiquitously involved in the organism and plays a key role in cellular energy metabolism. Metal ions can affect the NADH participating enzymes reactions. Aluminum ions (Al3+) are toxic to the nervous system and can cause a range of neurodegenerative diseases. Therefore, the study on the interaction between NADH and Al3+ is significantly helpful to understand the influence of Al3+ on the TCA cycle and enzymatic reaction in vivo. In this paper, the effects of Al3+ on the intrinsic fluorescence characteristics and molecular conformations of NADH in aqueous solutions have been investigated using UV-visible absorbance and steady-state fluorescence spectroscopy combined with time-correlated single photon counting technique (TCSPC). UV-visible absorption spectrum shows that NADH’s combination with Al3+ does not change the absorption characteristics of NADH’s adenine and reduced nicotinamide chromophores. We chose 340 nm as the excitation wavelength to avoid the fluorescence resonance energy transfer (FRET) between the NADH’s two intrinsic chromophores. It was proved that Al3+ could be coordinated to the two oxygen atoms on the hydroxyl group of NADH pyrophosphate bridge, which would make the NADH structure relatively more rigid, there by inhibiting the non-radiative processes such as the molecular rotation of NADH molecule in solution, resulting in the increase in the average fluorescence lifetime of the NADH molecule. Therefore, a fluorescence enhancement of the NADH molecule was observed by increasing Al3+ concentrations. Furthermore, we used the intrinsic fluorescence lifetimes amplitude ratio of NADH to characterize the two main conformational forms of the NADH in solution: folded form in which the adenine and nicotinamide groups are stacked parallel to each other, and unfolded form where the two bases apart from each other. It was found that Al3+ would break the original equilibrium of two conformations of NADH, making coenzyme NADH have a clear preference from the unfolded towards folded conformation, and eventually a new dynamic equilibrium would be achieved. Interestingly, it was revealed that the amplitude ratio increased logarithmically with the concentration of Al3+ when the concentration ratio of Al3+∶NADH was not more than 1∶2, showing a potential application prospect in Al3+ detection.