Improved Yield for the Enzymatic Synthesis of Radiolabeled Nicotinamide Adenine Dinucleotide

Abstract

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.

Figure 1

PPase improved the yield of the NMN adenylyl transferase reaction in vitro. (a) Proposed reaction for the enzymatic synthesis of ³²P-NAD⁺ from ³²P-ATP. Position of the ³²P label is indicated by a star. (b) NAD⁺, NADH, ATP, ADP, and AMP standards (>95% purity, 5 μL of 50 μM stocks) were resolved with thin layer chromatography (TLC) using PEI cellulose F plates and imaged following excitation at 254 nm. (c) Representative images of 1 μL from the enzymatic reactions being resolved with TLC and quantitated following exposure with a phosphorimaging screen. Leading edges were measured from the origins (dashed circles). (d) Calculated yields of ³²P-NAD⁺ (mean ± SD, n = 3, Student’s t test, *p < 0.01).

Figure 2

Selective uptake of radiolabeled reaction product by SLC25A51. (a) Representative ³²P dot blots of in vitro NAD⁺ import assays from DKO mitochondria that either expressed empty vector (EV) or a human mitochondrial NAD⁺ transporter (SLC25A51). (b) Relative mean intensities following uptake of ³²P product between paired EV and SLC25A51 conditions (mean ± SD, n = 4–5, Student’s t test, **p < 0.001).

Figure 3

Direct synthesis of labeled NADP from the ³²P-NAD⁺ reaction product. (a) Proposed reaction for the synthesis of labeled NADP from ³²P-NAD⁺. Position of the ³²P label is indicated by a star. (b) ADP, ATP, NAD⁺, and NADP standards were resolved with TLC using PEI cellulose F plates and imaged following excitation at 254 nm. (c) Samples (1 μL) from the NADK enzymatic reaction were obtained at indicated time points, resolved with TLC, and radioactivity was detected with phosphorimaging. Leading edges were measured from respective origins (dashed circles).