Fast and biphasic 8-nitroguanine production from guanine and peroxynitrite

Ana G Sánchez; M Natalia Ibargoyen; Mauricio Mastrogiovanni; Rafael Radi; Deborah J Keszenman; R Daniel Peluffo

doi:10.1016/j.freeradbiomed.2022.10.317

Fast and biphasic 8-nitroguanine production from guanine and peroxynitrite

Free Radic Biol Med. 2022 Nov 20;193(Pt 1):474-484. doi: 10.1016/j.freeradbiomed.2022.10.317. Epub 2022 Nov 2.

Authors

Ana G Sánchez¹, M Natalia Ibargoyen¹, Mauricio Mastrogiovanni², Rafael Radi², Deborah J Keszenman¹, R Daniel Peluffo³

Affiliations

¹ Grupo de Biofisicoquímica, Departamento de Ciencias Biológicas, CENUR Litoral Norte, Universidad de la República, Rivera 1350, 50000, Salto, Uruguay.
² Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, 11800, Montevideo, Uruguay.
³ Grupo de Biofisicoquímica, Departamento de Ciencias Biológicas, CENUR Litoral Norte, Universidad de la República, Rivera 1350, 50000, Salto, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Avda. General Flores 2125, 11800, Montevideo, Uruguay; Department of Pharmacology, Physiology and Neuroscience, Rutgers Biomedical and Health Sciences, New Jersey Medical School, Rutgers, The State University of New Jersey, 185 South Orange Avenue, Newark, NJ, 07103, USA. Electronic address: dpeluffo@unorte.edu.uy.

PMID: 36332879
DOI: 10.1016/j.freeradbiomed.2022.10.317

Abstract

Guanine (Gua), among purines, is a preferred oxidation/nitration target because of its low one-electron redox potential. The reactive oxygen/nitrogen species peroxynitrite (ONOO^-), produced in vivo by the reaction between nitric oxide (^•NO) and superoxide radical (O₂^•‒), is responsible for several oxidative modifications in biomolecules, including nitration, nitrosation, oxidation, and peroxidation. In particular, the nitration of Gua, although detected, as well as its reaction kinetics have been seldom investigated. Thus, we studied the concentration- and temperature-dependent formation of 8-nitroguanine (8-NitroGua) in phosphate buffer (pH 7.40) using stopped-flow spectrophotometry. Traces showed a biexponential behavior, with best-fit rate constants: k_fast = 4.4 s^-1 and k_slow = 0.41 s^-1 (30 °C, 400 μM both Gua and ONOO^-). k_fast increased linearly with the concentration of both reactants whereas k_slow was concentration-independent. Linear regression analysis of k_fast as a function of Gua and ONOO^- concentration yielded values of 2.5-6.3 × 10³ M^-1s^-1 and 1.5-3.5 s^-1 for the second-order (slope) and first-order (ordinate) rate constants, respectively (30 °C). Since ONOO^- is a short-lived species, its decay kinetics was also taken into account for this analysis. The 8-NitroGua product was stable for at least 4 h, so no spontaneous denitration was observed. Stopped-flow assays using antioxidants and free-radical scavengers suggested a mixed direct/indirect reaction mechanism for 8-NitroGua formation. Gua nitration by ONOO^- was also observed in the presence of physiologically relevant CO₂ concentrations. The reaction product identity, its yield (∼4.2%, with 400 μM ONOO^- and 200 μM Gua), and the reaction mechanism were unequivocally determined by HPLC-MS/MS experiments. In conclusion, 8-NitroGua production at physiologic pH reached significant levels in a few hundred milliseconds, suggesting that the process might be kinetically relevant in vivo and can likely cause permanent nitrative damage to DNA bases.

Keywords: Free radicals; Kinetics; Mass spectrometry; Nitration; Purines; Reactive nitrogen species; Stopped-flow.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Guanine / chemistry
Nitrates / chemistry
Nitric Oxide / chemistry
Peroxynitrous Acid*
Tandem Mass Spectrometry*

Substances

Peroxynitrous Acid
8-nitroguanine
Nitrates
Guanine
Nitric Oxide