title: High-throughput, homogeneous, fluorescence intensity-based measurement of adenosine diphosphate and other ribonucleoside diphosphates with nanomolar sensitivity.
abstract: A new, homogeneous, high-throughput-compatible assay method is described for the fluorescence-based quantitation of nanomolar concentrations of ribonucleoside diphosphates (rNDPs). The principle of the method is the conversion of the rNDPs to RNA by the enzyme polynucleotide phosphorylase (EC 2.7.7.8) and detection of the RNA by the increased fluorescence of a commercial nucleic acid detection dye. A commercial RNA homopolymer complementary to the RNA product is included to increase the sensitivity for ADP and UDP. Standard curves for nanomolar concentrations of ADP, UDP, GDP, and CDP are shown. The assay detected 75 nM ADP produced by the pyruvate kinase-catalyzed phosphorylation of pyruvate with a signal-to-baseline ratio of 2.8. The assay may be used in either a continuous or a discontinuous mode.
authors.affiliation: Bioscience Department, Infection Innovative Medicines, AstraZeneca R&D Boston, Waltham, MA 02451, USA. adam.shapiro@astrazeneca.com
authors.forename: Adam B
authors.initials: AB
authors.lastname: Shapiro
authors.forename: Laurel
authors.initials: L
authors.lastname: Hajec
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
mesh_terms: D000244:Adenosine Diphosphate / Q000032:analysis*; D003565:Cytidine Diphosphate / Q000032:analysis; D005456:Fluorescent Dyes / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000032:analysis; D057166:High-Throughput Screening Assays / Q000379:methods; D007700:Kinetics; D010766:Phosphorylation; D011770:Pyruvate Kinase / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014530:Uridine Diphosphate / Q000032:analysis
chemical_list: D005456:Fluorescent Dyes; D011773:Pyruvates; D006153:Guanosine Diphosphate; D014530:Uridine Diphosphate; D000244:Adenosine Diphosphate; D003565:Cytidine Diphosphate; D012313:RNA; D011770:Pyruvate Kinase