Abstract
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 1 m K 2HPO 4:10% 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.
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