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 (oxo(8)GTP), 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 Cu(2+) 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 oxo(8)GTP 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 Cu(2+) 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.

Figure 1. EPR spectra of GTP, L-ascorbic acid, and Cu(II)

A) and B) EPR spectra of control experiments in PBS, pH =7.4, 1 mM L-ascorbic acid, and 80mM DMPO incubated for 2 min (A) and 30 min (B). C) to H) EPR spectra of reactions of 1 mM GTP, 1 mM L-ascorbic acid, 10 μM Cu(II) sulfate, and 80mM DMPO in air-saturated PBS, pH 7.4 solution. C′) to H′) EPR spectra of reactions exactly as in C to H, with the exception that the PBS pH 7.4 solution is devoid of oxygen by saturation with argon for 30 minutes. All spectra were collected at room temperature (22 °C) and plotted on the same scale (magnetic field and intensity of EPR) for comparison. EPR conditions: receiver gain 5.02 × 10⁴, modulation amplitude 1 G, time constant 163.84 ms, frequency 9.687 GHz, power 20 mW and 1024 points resolution.

Figure 2. EPR spectra of GTP, L-ascorbic acid, and Cu(II) with DMSO

Experiment conducted in PBS, pH =7.4, 1mM L-ascorbic acid, and 80 mM DMPO incubated for 1 min (A) or 3 min (B). EPR conditions: receiver gain 5.02 × 10⁴, modulation amplitude 1 G, time constant 163.84 ms, frequency 9.687 GHz, power 20 mW and 1024 points resolution.

Figure 3. EPR spectra of GTP and Cu(II) or L-ascorbic acid

A) to G) EPR spectra of the reaction of 1mM GTP, 10 μM Cu(II) sulfate, and 80 mM DMPO in air-saturated PBS, pH=7.4 solution. H) to L) EPR spectra of the reaction of 1 mM GTP, 1 mM L-ascorbic acid, and 80 mM DMPO in air-saturated PBS, pH 7.4 solution. M) to P) EPR spectra of the reaction of 1mM L-ascorbic acid, 10 μM Cu(II) sulfate, and 80 mM DMPO in air-saturated PBS, pH=7.4 solution. All spectra were collected at room temperature (22 °C) and plotted on the same scale (magnetic field and intensity of EPR) for comparison. EPR conditions: receiver gain 5.02 × 10⁴, modulation amplitude 1 G, time constant 163.84 ms, frequency 9.687 GHz, power 20 mW and 1024 points resolution.

Figure 4. Effects of pH and reactant concentrations on free radical generation

A) and N) EPR spectra of 1 mM L-ascorbic and 80 mM DMPO in PBS, pH 7.0 (A) and PBS, pH 5.7 (N); B) to E) EPR spectra of the reaction of 1 mM GTP, 1 mM L-ascorbic acid, 10 μM Cu(II) sulfate, and 80 mM DMPO, in air-saturated PBS, pH 7.0 solution. F) to I) EPR spectra of the reaction of 2 mM GTP, 1 mM L-ascorbic acid, 30 μM Cu(II) sulfate, and 80 mM DMPO, in air-saturated PBS, pH=7.0 solution. J) to M) EPR spectra of the reaction of 3 mM GTP, 1mM L-ascorbic acid, 40 μM Cu(II) sulfate, and 80 mM DMPO, in air-saturated PBS, pH 7.0 solution. O) to P) EPR spectra of the reaction of 1 mM GTP, 1 mM L-ascorbic acid, 10 μM Cu(II) sulfate, and 80 mM DMPO, in air-saturated PBS, pH 5.7 solution. All spectra were collected at room temperature (22 °C) and plotted on the same scale (magnetic field and intensity of EPR) for comparison. EPR conditions: receiver gain 5.02 × 10⁴, modulation amplitude 1 G, time constant 163.84 ms, frequency 9.687 GHz, power 20 mW and 1024 points resolution.

Figure 5. Direct EPR analysis of Cu(II) and GTP

A) EPR spectrum of 2 mM Cu(II) sulfate. B) EPR spectrum of 500 μM Cu(II) sulfate with 2 mM of GTP. All solutions diluted in water, PBS, and 5% glycerol. EPR conducted at 77K.

Figure 6. Oxygen consumption by GTP, Cu(II), and L-ascorbic acid

Oxygen measurements using a Clark electrode of the reaction of 1 mM GTP, 1 mM L-ascorbic acid, 10 μM Cu(II) sulfate (▪) or Cu(Imine) (), in air-saturated PBS, pH 7.4 solution in a total cell volume of 1.5 mL at (22.0 ± 0.2) °C.

Figure 7. Oxo⁸GTP formation analysis by HPLC-EC and MALDI-TOF

A) and D) are HPLC-EC chromatogram and MALDI-TOF spectra of oxo⁸G (reported as oxo⁸GTP, see Methods) and GTP respectively, in a four-hour incubation of 1 mM GTP and 1 mM L-ascorbic acid at 37°C. Inset of D) is from a standard solution containing 1:1 GTP:Oxo⁸GTP (m/z GTP:522.21; m/z oxo⁸GTP: 539.41). B) and E) is a four-hour incubation of 1mM GTP and 200 μM Cu(II) sulfate at 37°C. C) and F) is a four-hour incubation of 1mM GTP, 1mM L-ascorbic acid, and 10 μM Cu(II) sulfate at 37°C. HPLC-EC chromatograms show traces collected at 250 mV EC channel. MALDI-TOF spectra are normalized to % Intensity of signal and baselines were corrected for noise.

Figure 8. Oxo⁸GTP in GTP, Cu(II), L-ascorbic acid and Aβ_1-42 reactions

A) Oxo⁸GTP levels quantified by HPLC-EC in one-hour incubations of 1mM GTP and increasing concentrations of L-ascorbic acid. B) Oxo⁸GTP levels quantified by HPLC-EC in four-hour incubations of 1 mM GTP, 1 mM L-ascorbic acid, and increasing concentrations of Cu(II) sulfate. C) Oxo⁸GTP levels quantified by HPLC-EC in four-hour incubations of 1 mM GTP and 1 mM L-ascorbic acid, 200 μM Cu(II) sulfate, 1mM L-ascorbic acid/ 10 μM Cu(II) sulfate, 100 nM Aβ_1-42, and 10 μM Cu(II) sulfate/100 nM Aβ_1-42 at 37°C. Data represent mean ± SEM. n= 2-9. * p< 0.05 applying a one-way ANOVA with Newman-Keuls Multiple Comparison Test post-hoc analysis.

Figure 9. Mechanism proposed for the oxidation of GTP by Cu(II) and L-ascorbic acid