A general procedure is described for developing and validating HPLC-based assays for nucleotide-utilizing enzymes. The method is comparable in sensitivity to spectrophotometric, enzyme-coupled assays and is applicable to any enzymatic reaction involving coenzyme A and its esters, or the common nucleoside tri-, di-, and/or monophosphates. Nucleotide products of the enzymatic reactions are directly quantitated from their uv absorbances. A typical HPLC-based assay involves running an enzymatic reaction for a fixed time, stopping the reaction by adding acid, and using HPLC to quantitate the amount(s) of nucleotide reactant(s) converted to product(s). Nucleotides containing the same base can be separated under isocratic conditions using a 5-μm C-18, reversed-phase column, and a mobile phase at pH 5 containing 100 mM potassium phosphate, 20 mM acetate, 5 mM tetrabutylammonium ion as an ion-pairing agent, and sufficient acetonitrile to keep the run time within 10 min. Separation of nucleotides containing different bases can usually be accomplished under isocratic conditions by the proper choice of the pH within the range of 3 to 7. A microcomputer is used to control all system hardware and to automate collection and processing of chromatographic data. Application of the general method to development of assays for nucleoside diphosphate kinase and succinyl-CoA synthetase is described. The assay for nucleoside diphosphate kinase quantitates UTP formed from UDP and ATP as substrates. The assay for succinyl-CoA synthetase measures succinyl-CoA after HPLC separation of reactants and products at pH 4 where succinyl-CoA is stable to chemical hydrolysis. © 1993 Academic Press. All rights reserved.
