Aix Marseille Univ, CNRS, Centrale Marseille, iSm2, Marseille, France.
INRAE, Aix Marseille Univ, UMR1163 Biodiversité et Biotechnologie Fongiques, 13009, Marseille, France.
Analyst. 2022 May 30;147(11):2515-2522. doi: 10.1039/d2an00200k.
1D H NMR spectroscopy has been widely used to monitor enzymatic activity by recording the evolution of the spectra of substrates and/or products, thanks to the linear response of NMR. For complex systems involving the coexistence of multiple compounds (substrate, final product and various intermediates), the identification and quantification can be a more arduous task. Here, we present a simple analytical method for the rapid characterization of reaction mixtures involving enzymatic complexes using Maximum Quantum (MaxQ) NMR, accelerated with the Non-Uniform Sampling (NUS) acquisition procedure. Specifically, this approach enables, in the first analytical step, the counting of the molecules present in the samples. We also show, using two different enzymatic systems, that the implementation of these pulse sequences implies precautions related to the short relaxation times due to the presence of metallo-enzymes or paramagnetic catalysts. Finally, the combination of MaxQ and diffusion experiments, which leads to a 3D chart, greatly improves the resolution and offers an extreme simplification of the spectra while giving valuable indications on the affinity of the enzymes to the different compounds present in the reaction mixture.
1D H NMR 光谱学已被广泛用于通过记录底物和/或产物的光谱演变来监测酶活性，这要归功于 NMR 的线性响应。对于涉及多种化合物（底物、最终产物和各种中间产物）共存的复杂系统，鉴定和定量可能是一项更加艰巨的任务。在这里，我们提出了一种使用最大量子（MaxQ）NMR 对涉及酶复合物的反应混合物进行快速特征分析的简单分析方法，该方法通过非均匀采样（NUS）采集程序进行加速。具体来说，这种方法能够在第一个分析步骤中对样品中存在的分子进行计数。我们还使用两种不同的酶系统表明，这些脉冲序列的实现需要注意由于存在金属酶或顺磁催化剂而导致的短弛豫时间。最后，MaxQ 和扩散实验的组合产生了一个 3D 图谱，大大提高了分辨率，并在提供有关酶对反应混合物中不同化合物亲和力的有价值信息的同时，使光谱极端简化。
