We measured the production of reactive hydroxyl radical (<tex-math>${\rm OH}^{\bullet}$</tex-math>) by Fe2+ itself or complexed with nucleotide triphosphates or tripolyphosphate (TPP). Coumarin-3-carboxylic acid (3-CCA) reacts with the <tex-math>${\rm OH}^{\bullet}$</tex-math> produced by <tex-math>${\rm Fe}^{2+},{\rm Fe}^{3+}$</tex-math> or Cu2+ plus ascorbate and with various iron complexes. We measured in real time the increased fluorescence of 3-CCA after hydroxylation to 7-hydroxy-coumarin-3-carboxylic acid (7-OHCCA). Phosphate-buffered solutions do not affect the yield of Fe2+-linked <tex-math>${\rm OH}^{\bullet}$</tex-math> as do other organic buffer solutions. Our results show that guanosine triphosphate enhances the Fe2+-linked production of <tex-math>${\rm OH}^{\bullet}$</tex-math>. We also tested inosine triphosphate, adenosine triphosphate and xanthine triphosphate for their capacity to produce <tex-math>${\rm OH}^{\bullet}$</tex-math> with Fe2+. Inosine triphosphate is the most effective nucleotide in the production of <tex-math>${\rm OH}^{\bullet}$</tex-math>. However, the Fe2+-mediated yield of <tex-math>${\rm OH}^{\bullet}$</tex-math> is greater in the presence of TPP compared to the nucleotide triphosphates. Organic buffers as well as the purine and ribose portion of nucleotides compete for <tex-math>${\rm OH}^{\bullet}$</tex-math> and decrease the yield of fluorescent 7-OHCCA. We also decreased the yield of <tex-math>${\rm OH}^{\bullet}$</tex-math> by adding guanosine to the Fe2+/TPP-generating system. Adenosine, ribose and deoxyribose also react with Fe2+-generated <tex-math>${\rm OH}^{\bullet}$</tex-math>. The decreased yield of 7-OHCCA occurs because the ribose and purine part of the molecule reacts with <tex-math>${\rm OH}^{\bullet}$</tex-math>. The maximal production of reactive <tex-math>${\rm OH}^{\bullet}$</tex-math>, compared to all nucleotides and phosphates tested, occurs with a ratio of 2 <tex-math>${\rm TPP}/{\rm Fe}^{2+}$</tex-math> complex. In conclusion, the real-time measurement of the production of fluorescent 7-OHCCA provides a convenient means for measuring chemically generated <tex-math>${\rm OH}^{\bullet}$</tex-math>. The <tex-math>${\rm TPP}/{\rm Fe}^{2+}$</tex-math>-generating mixture, in the presence of 3-CCA, can be used to study the scavenging ability of other competing molecules.

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