High-performance liquid chromatography-based assays of enzyme activities

Interest in using HPLC to assay enzymatic reactions continues to grow as evidenced by the more than 100 papers published during the early 1990s. HPLC can be used for any enzymatic assay that requires separation of substrates and products before quantifying the extent of the reaction. The popularity of HPLC-based assays is due to several reasons: (1) HPLC offers unsurpassed precision, specificity, sensitivity, and reproducibility. (2) Powerful microcomputers and user-friendly software automate the running of samples and collection and processing of data. (3) Current columns, especially C18 packings, separate a very wide variety of samples, and (4) A variety of on-line detectors provide a means to detect virtually any compound. This review surveys recent papers on the development of HPLC-based assays for enzymes that degrade or otherwise modify macromolecules. Methods for assaying enzymes involved in metabolic pathways are also reviewed. Work by the authors in developing HPLC-based assays for mitochondrial enzymes that use GTP/GDP and other nucleotides that cannot be or are not easily assayed by enzyme-coupled assays is discussed. These enzymes include nucleoside diphosphate kinase, succinate thiokinase, and GTP—AMP phospho-transferase. The assays are suitable for determining the submitochondrial compartmentation of enzyme activities. Finally, current and anticipated trends in HPLC technology, including new column packings and the trend toward smaller columns that give faster separations, are reviewed in relation to enzyme assays.