Development of an HPLC-based guanosine monophosphate kinase assay and application to Plasmodium vivax guanylate kinase.

ABSTRACT: The development of a high-performance liquid chromatography (HPLC)-based method, for guanosine monophosphate kinase activity assays, is presented. The method uses the intrinsic UV absorption (at 260 nm) of substrates and products of the enzymatic reaction (GMP, ATP, ADP and GDP) to unambiguously determine percent conversion of substrate into product. It uses a commercially available C18 column which can separate reaction samples by elution under isocratic conditions in 12 min per run. The kinetics of the forward reaction catalyzed by Plasmodium vivax guanylate kinase (PvGK), a potential drug target against malaria, was determined. The relative concentrations of the two substrates (GMP and ATP) have a distinct effect on reaction velocity. Kinetic analyses showed the PvGK-catalyzed reaction

SUBMITTER: Pedro L 

PROVIDER: S-EPMC6494078 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature