Quantitation of Hydroxyl Radicals Produced by Radiation and Copper-Linked Oxidation of Ascorbate by 2-Deoxy-d-Ribose Method

We have established controlled conditions for studying the reaction of chemically and radiolytically produced hydroxyl radical (OH) with 2-deoxy-D-ribose (2-DR). Ascorbate (ASC) or dithiothreitol (DTT) and cuprous or cupric ions were used to generate the OH-radical. The OH-radical was detected using the classical method of measuring the amount of thiobarbituric acid reactive products (TBARP) formed by OH-mediated 2-DR degradation, but using sensitive fluorescent detection of the TBARP production to quantify the OH-radical. All experiments were performed with adequate O₂ concentrations. The copper reaction with ASC consumes O₂ in a manner that is strongly dependent on copper concentration, and less dependent on ascorbate concentration. For an independent check of the Cu²⁺ catalyzed ASC oxidation kinetics, the decay of ASC absorbency at 265 nm, as well as the increase of H₂O₂ absorbency at 240 nm, were also monitored. These spectral changes agree well with the O₂ consumption data. TBARP production from 2-DR incubated with a Cu²⁺–ASC mixture or γ-irradiated were also compared. γ-Irradiation of 2-DR solutions shows a dose and 2-DR concentration dependent increase of TBARP generation. Other electron donors, such as DTT, are more complicated in their mechanism of OH-radical production. Incubation of 2-DR with Cu²⁺-DTT mixtures shows a delay (50 min) before OH-radical generation is detected. Our results suggest that the Cu²⁺-ASC reaction can be used to mimic the effects of ionizing radiation with respect to OH-radical generation. The good reproducibility and relative simplicity of the 2-DR method with fluorescence detection indicates its usefulness for the quantitation of the OH-radical generated radiolytically or chemically in carefully controlled model systems. © 1997 Elsevier Science Inc.