title: A colorimetric assay for the quantitation of free adenine applied to determine the enzymatic activity of ribosome-inactivating proteins.
abstract: Adenine quantitation is required for a variety of applications. To date, the prevalent method for quantifying free adenine, in a variety of applications, is the detection of fluorescent-derivatized adenine by HPLC. For the present study, we developed a high-throughput, nonradioactive, enzyme-based colorimetric adenine quantitation assay that is performed in one multireaction incubation step. The assay does not require adenine derivatization and is designed for microplates. The key step is the conversion of adenine to adenosine monophosphate by adenine phosphoribosyl transferase. Subsequent reactions finally produce three inorganic phosphate ions per adenine molecule. Phosphate is quantitated by the color-generating phosphorylysis of a particular purine derivate. Ribosome-inactivating proteins that release adenine from polynucleotides are often used to investigate intracellular protein trafficking and are important for the design of immunotoxins. We therefore used ricin, dianthin, saporin, and a variety of saporin fusion proteins to show that this method is suitable for quantifying adenine release using different substrates. The measured rate of adenine release and substrate specificity are comparable to those determined by HPLC and radioactive detection techniques.
authors.affiliation: Institut für Klinische Chemie und Pathobiochemie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, Berlin, D-12200, Germany.
authors.forename: Iring
authors.initials: I
authors.lastname: Heisler
authors.forename: Jutta
authors.initials: J
authors.lastname: Keller
authors.forename: Rudolf
authors.initials: R
authors.lastname: Tauber
authors.forename: Mark
authors.initials: M
authors.lastname: Sutherland
authors.forename: Hendrik
authors.initials: H
authors.lastname: Fuchs
mesh_terms: D000225:Adenine / Q000032:analysis*; D001681:Biological Assay; D003124:Colorimetry / Q000379:methods; D000922:Immunotoxins; D007700:Kinetics; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000032:analysis*; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes; D012276:Ricin / Q000032:analysis*; D000076985:Saporins; D012680:Sensitivity and Specificity
chemical_list: D000922:Immunotoxins; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D012276:Ricin; D009699:N-Glycosyl Hydrolases; D000076985:Saporins; D000225:Adenine