Improved Yield for the Enzymatic Synthesis of Radiolabeled Nicotinamide Adenine Dinucleotide.
Improved Yield for the Enzymatic Synthesis of Radiolabeled Nicotinamide Adenine Dinucleotide.
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DOI：
10.1021/acsbiomedchemau.2c00065
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发表时间：
2023-02-15
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影响因子：
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通讯作者：
Cambronne, Xiaolu A
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文献类型：
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作者：
Eller, Jared;Goyal, Shivansh;Cambronne, Xiaolu A
标记的β-烟酰胺腺嘌呤二核苷酸（NAD）类似物对于发现新的生物化学连接和定量酶活性至关重要。它们作为示踪剂用于酶学、通量分析和原位测量。然而，特定类型的类似物，特别是放射性标记的NAD同位素体的可用性有限。在这里，我们描述了一种改进的32 P-NAD+的酶促合成反应，产率为98% ± 1%，使用降低的反应物浓度和标准设备。这代表了迄今为止酶促合成NAD+的最高报道产率。由于产率高，我们能够直接使用反应产物来生成衍生物，例如32 P-NADP。高产量的酶促合成对于各种各样的标记物和NAD衍生物是通用的。其优点包括降低反应物浓度，为下游应用提供足够量的产物，并最大限度地减少中间纯化步骤。
Labeled β-nicotinamide adenine dinucleotide (NAD) analogues have been critical for uncovering new biochemical connections and quantitating enzymatic activity. They function as tracers for enzymology, flux analyses, and in situ measurements. Nevertheless, there is limited availability of specific types of analogues, especially radiolabeled NAD isotopologues. Here, we describe an improved enzymatic synthesis reaction for 32P- NAD+ with a yield of 98% ± 1%, using lowered concentrations of reactants and standard equipment. This represents the highest reported yield for the enzymatic synthesis of NAD+ to date. With the high yield we were able to directly use the reaction product to generate derivatives, such as 32P-NADP. The high-yield enzymatic synthesis is versatile for a broad variety of labels and NAD derivatives. Its advantages include lowered concentrations of reactants, providing sufficient amounts of product for downstream applications, and minimizing intermediate purification steps.