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首页> 外文期刊>The journal of physical chemistry, B. Condensed matter, materials, surfaces, interfaces & biophysical >A Novel Tri-Enzyme System in Combination with Laser-Driven NMR Enables Efficient Nuclear Polarization of Biomolecules in Solution
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A Novel Tri-Enzyme System in Combination with Laser-Driven NMR Enables Efficient Nuclear Polarization of Biomolecules in Solution

机译：结合激光驱动的NMR的新型三酶系统可实现溶液中生物分子的有效核极化。

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摘要

NMR is an extremely powerful, yet insensitive technique. Many available nuclear polarization methods that address sensitivity are not directly applicable to low-concentration biomolecules in liquids and are often too invasive. Photochemically induced dynamic nuclear polarization (photo-ClDNP) is no exception. It needs high-power laser irradiation, which often leads to sample degradation, and photosensitizer reduction. Here, we introduce a novel tri-enzyme system that significantly overcomes the above challenges, rendering photo-CIDNP a practically applicable technique for NMR sensitivity enhancement in solution. The specificity of the nitrate reductase (NR) enzyme is exploited to selectively in situ reoxidize the reduced photo-CIDNP dye FMNH2. At the same time, the oxygen-scavenging ability of glucose oxidase (GO) and catalase (CAT) is synergistically employed to prevent sample photodegradation. The resulting tri-enzyme system (NR-GO-CAT) enables prolonged sensitivity-enhanced data collection in ID and 2D heteronuclear NMR, leading to the highest photo-CIDNP sensitivity enhancement (48-fold relative to SE-HSQC) achieved to date for amino acids and polypeptides in solution. NR-GO-CAT extends the concentration limit of photo-CIDNP NMR down to the low micromolar range. In addition, sensitivity (relative to the reference SE-HSQC) is found to be inversely proportional to sample concentration, paving the way for the future analysis of even more diluted samples.
机译：NMR是一种非常强大但不敏感的技术。解决敏感度问题的许多可用的核极化方法并不直接适用于液体中的低浓度生物分子，并且往往具有侵入性。光化学诱导的动态核极化（photo-ClDNP）也不例外。它需要大功率的激光照射，这通常会导致样品降解和光敏剂减少。在这里，我们介绍了一种新型的三酶系统，该系统可以极大地克服上述挑战，从而使photo-CIDNP成为提高溶液中NMR灵敏度的实用技术。利用硝酸还原酶（NR）酶的特异性来选择性地原位还原被还原的光CIDNP染料FMNH2。同时，协同使用葡萄糖氧化酶（GO）和过氧化氢酶（CAT）的除氧能力，以防止样品光降解。所得的三酶系统（NR-GO-CAT）可以延长ID和2D异核NMR中的灵敏度增强的数据收集，从而实现了迄今为止迄今为止最高的光CIDNP灵敏度增强（相对于SE-HSQC是48倍）溶液中的氨基酸和多肽。 NR-GO-CAT将光CIDNP NMR的浓度极限扩展到低微摩尔范围。此外，发现灵敏度（相对于参考SE-HSQC）与样品浓度成反比，为将来分析更多稀释的样品铺平了道路。

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