Hydroxyl radical oxidation of guanosine 5′-triphosphate (GTP): requirement for a GTP–Cu(II) complex

Abstract

Levels of oxidized guanosine base in DNA have become a hallmark biomarker in assessing oxidative stress implicated in a variety of disease and toxin-induced states. However, there is evidence that the guanosine in the nucleotide triphosphate pool (GTP) is more susceptible to oxidation than guanosine residues incorporated into nucleic acids and this causes a substantial amount of the oxidized product, 8-oxoguanosine 5′-triphosphate (oxo⁸GTP), to accumulate in cell-free and in cell-culture preparations. Electron paramagnetic resonance (EPR) spectroscopy and direct EPR analysis of free radical production by copper sulfate and L-ascorbic acid demonstrates that the hydroxyl radical (HO^•) is produced via oxidation of Cu⁺ to Cu²⁺ while in a complex with GTP. This HO^• production is dependent on the availability of oxygen and the presence of GTP in the reaction milieu. Verification of free radical-mediated production of oxo⁸GTP is presented using HPLC with electrochemical detection and matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry (MALDI-LTOF-MS). The sum of these results is presented in a novel mechanism of GTP oxidation by Cu²⁺ and L-ascorbic acid. A better understanding of the chemistry involved in this oxidative modification of GTP facilitates a more comprehensive understanding of its potential physiological consequences.

Keywords:

• OXIDATIVE STRESS
• FREE RADICALS
• GUANOSINE TRIPHOSPHATE
• GTP
• GUANOSINE
• HYDROXYL RADICAL
• COPPER
• ASCORBATE