Characterization of oxidized nicotinamide adenine dinucleotide (NAD+) analogues using a high-pressure-liquid-chromatography-based NAD+-glycohydrolase assay and comparison with fluorescence-based measurements

A high-pressure-liquid-chromatography (HPLC)-based technique was developed to assess the oxidized nicotinamide adenine dinucleotide (NAD+)-glycohydrolase activity of the catalytic domain of Pseudomonas exotoxin A containing a hexa-His tag. The assay employs reverse-phase chromatography to separate the substrate (NAD+) and products (adenosine 5′-diphosphate-ribose and nicotinamide) produced over the reaction time course, whereby the peak area of nicotinamide is correlated using a standard curve. This technique was used to determine whether the NAD+ analogue, 2′-F-ribo-NAD+, was a competing substrate or a competitive inhibitor for this toxin. This NAD+ analogue was hydrolyzed at a rate of 0.2% that of NAD+ yet retained the same binding affinity for the toxin as the parent compound. Finally, the rate that a fluorescent NAD+ analogue, ε-NAD+, is hydrolyzed by the toxin was also investigated. This analogue was hydrolyzed six times slower than NAD+ as determined using HPLC. The rate of hydrolysis of ε-NAD+ calculated using the fluorometric version of the assay shows a sixfold increase in reaction rate compared to that determined by HPLC. This HPLC-based assay is adaptable to any affinity-tagged enzyme that possesses NAD+-glycohydrolase activity and offers the advantage of directly measuring the enzyme-catalyzed hydrolytic rate of NAD+ and its analogues.