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 chromtography (HPLQ-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, epsilon-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 epsilon-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. (c) 2005 Elsevier Inc. All rights reserved.