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| Fluorescence Enhancement and Conformational Studies of Coenzyme NADH With Aluminum |
| ZHANG Meng-jie, CAO Si-min, WANG Meng-yu, LI Hao-yang, LI Dong, ZHAO Ze-nan, XU Jian-hua* |
| State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai 200241, China |
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Abstract 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.
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Received: 2020-04-10
Accepted: 2020-07-15
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Corresponding Authors:
XU Jian-hua
E-mail: jhxu@phy.ecnu.edu.cn
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