title: A continuous spectrophotometric enzyme-coupled assay for deoxynucleoside triphosphate triphosphohydrolases.
abstract: We describe a continuous, spectrophotometric, enzyme-coupled assay useful to monitor reactions catalyzed by nucleoside triphosphohydrolases. In particular, using Escherichia coli deoxynucleoside triphosphohydrolase (Dgt), which hydrolyzes dGTP to deoxyguanosine and tripolyphosphate (PPPi) as the enzyme to be tested, we devised a procedure relying on purine nucleoside phosphorylase (PNPase) and xanthine oxidase (XOD) as the auxiliary enzymes. The deoxyguanosine released by Dgt can indeed be conveniently subjected to phosphorolysis by PNPase, yielding deoxyribose-1-phosphate and guanine, which in turn can be oxidized to 8-oxoguanine by XOD. By this means, it was possible to continuously detect Dgt activity at 297 nm, at which wavelength the difference between the molar extinction coefficients of 8-oxoguanine (8000 M(-1) cm(-1)) and guanine (1090 M(-1) cm(-1)) is maximal. The initial velocities of Dgt-catalyzed reactions were then determined in parallel with the enzyme-coupled assay and with a discontinuous high-performance liquid chromatography (HPLC) method able to selectively detect deoxyguanosine. Under appropriate conditions of excess auxiliary enzymes, the activities determined with our continuous enzyme-coupled assay were quantitatively comparable to those observed with the HPLC method. Moreover, the enzyme-coupled assay proved to be more sensitive than the chromatographic procedure, permitting reliable detection of Dgt activity at low dGTP substrate concentrations.
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
authors.forename: Deepa
authors.initials: D
authors.lastname: Singh
authors.affiliation: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
authors.forename: Roel M
authors.initials: RM
authors.lastname: Schaaper
authors.affiliation: Department of Pharmacy and Biotechnology, University of Bologna, 40136 Bologna, Italy; CSGI, University of Firenze, 50019 Sesto Fiorentino, FI, Italy. Electronic address: a.hochkoeppler@unibo.it.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Hochkoeppler
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology; D043583:Nucleoside-Triphosphatase / Q000032:analysis*; D011683:Purine-Nucleoside Phosphorylase / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D014969:Xanthine Oxidase / Q000032:analysis
chemical_list: D014969:Xanthine Oxidase; D011683:Purine-Nucleoside Phosphorylase; D000469:Alkaline Phosphatase; D043583:Nucleoside-Triphosphatase
keywords: 8-Oxoguanine; Deoxynucleoside triphosphatase; Enzyme-coupled assay; Escherichia coli Dgt; Purine nucleoside phosphorylase; Xanthine oxidase