Analysis of NAD+ in picomole amounts with sodium [32P]pyrophosphate and NAD-pyrophosphorylase.

A new method is described for the determination of NAD⁺ in picomole amounts. An enzymatic coupling system of NAD-pyrophosphorylase and hexokinase is used to convert sodium ³²P]pyrophosphate and NAD⁺ to ³²P]ADP, glucose 6-³²P]phosphate, and NMN. The key step in this analysis is the selective adsorption of the reaction product ³²P]ADP, onto activated charcoal with a solution of 1m K₂HPO₄:10% trichloroacetic acid (1:3, v/v, pH 2). The range of concentrations of NAD⁺ that can be measured is 1–200 pmol. The simplicity of the method allows as many as 180 samples to be assayed in 4–5 h. This procedure has been used to quantitate NAD⁺ in crude extracts of germinating wheat embryos.