Analysis of Phosphodiesterase Reaction Mixtures by High-Performance Liquid Chromatography

This chapter discusses a few technical updates and one specific example using simple HPLC procedures for the analysis of phosphodiesterase reaction mixtures. Ion-exchange HPLC columns, in contrast to reversed-phase columns, have been the columns of choice for enzymes such as adenylate cylase and phosphodiesterase because of their ability to resolve easily the various nucleotides in a simple and reproducible manner. Columns of different sizes are available from a variety of vendors. Reproducible and simple systems that use 4.6-mm-diameter columns are currently available with detailed protocols. The details of the system depend on the HPLC hardware that is available in the laboratory. The commercial availability of prepacked columns and protocols makes the set-up adaptable and easy to use. The ability to resolve metabolites and inhibitors and the rapid, unattended fractionation and quantification of components in enzyme-catalyzed reaction mixtures make it applicable to a variety of uses in the study of cyclic nucleotide metabolism.