title: The rapid and direct determination of ATPase activity by ion exchange chromatography and the application to the activity of heat shock protein-90.
abstract: Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2mm×6mm i.d.), under a three-solvent gradient elution mode and UV detection at 256nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples.
authors.affiliation: Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6, 40126 Bologna, Italy. manuela.bartolini3@unibo.it
authors.forename: Manuela
authors.initials: M
authors.lastname: Bartolini
authors.forename: Irving W
authors.initials: IW
authors.lastname: Wainer
authors.forename: Carlo
authors.initials: C
authors.lastname: Bertucci
authors.forename: Vincenza
authors.initials: V
authors.lastname: Andrisano
mesh_terms: D000244:Adenosine Diphosphate / Q000032:analysis; D000249:Adenosine Monophosphate / Q000032:analysis; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000032:analysis; D002138:Calibration; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004789:Enzyme Activation; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D006868:Hydrolysis; D057230:Limit of Detection
chemical_list: D018841:HSP90 Heat-Shock Proteins; C533585:HSP90AA2P protein, human; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases