Direct real-time evaluation of nitration with green fluorescent protein in solution and within human cells reveals the impact of nitrogen dioxide vs. peroxynitrite mechanisms

Abstract

3-Nitrotyrosyl adducts in proteins have been detected in a wide range of diseases. The mechanisms by which reactive nitrogen oxide species may impede protein function through nitration were examined by using a unique model system, which exploits a critical tyrosyl residue in the fluorophoric pocket of recombinant green fluorescent protein (GFP). Exposure of purified GFP suspended in phosphate buffer to synthetic peroxynitrite in either 0.5 or 5 M steps resulted in progressively increased 3-nitrotyrosyl immunoreactivity concomitant with disappearance of intrinsic fluorescence (IC 50 Ϸ 20 M). Fluorescence from an equivalent amount of GFP expressed within intact MCF-7 tumor cells was largely resistant to this bolus treatment (IC 50 > 250 M). The more physiologically relevant conditions of either peroxynitrite infusion (1 M͞min) or de novo formation by simultaneous, equimolar generation of nitric oxide (NO) and superoxide (e.g., 3-morpholinosydnonimine; NONOates plus xanthine oxidase͞hypo-xanthine, menadione, or mitomycin C) were examined. Despite robust oxidation of dihydrorhodamine under each of these conditions, fluorescence decrease of both purified and intracellular GFP was not evident regardless of carbon dioxide presence, suggesting that oxidation and nitration are not necessarily coupled. Alternatively, both extra-and intracellular GFP fluorescence was exquisitely sensitive to nitration produced by heme-peroxidase͞hydrogen peroxidecatalyzed oxidation of nitrite. Formation of nitrogen dioxide (NO 2) during the reaction between NO and the nitroxide 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide indicated that NO 2 can enter cells and alter peptide function through tyrosyl nitration. Taken together, these findings exemplified that heme-peroxidasecatalyzed formation of NO 2 may play a pivotal role in inflammatory and chronic disease settings while calling into question the significance of nitration by peroxynitrite.3-nitrotyrosine ͉ myeloperoxidase ͉ nitric oxide ͉ xanthine oxidase ͉ superoxide T he prevalence of increased 3-nitrotyrosine in both acute inflammatory events and numerous chronic diseases suggests that protein nitration may be an important component in pathophysiologic processes (1-7). However, there is disagreement regarding the predominant mechanism by which tyrosyl nitration occurs under these conditions (1-16). The reaction between superoxide (O 2 Ϫ ) and nitric oxide (NO) results in formation of peroxynitrite (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)17). Numerous studies have concluded that this nitrogen oxide mediates the majority of nitration, on the basis of 3-nitrotyrosine formation after bolus application of synthetically produced peroxynitrite. Nitration by this route is strongly influenced by buffer composition, in particular, the concentration of protons, transition metals, and carbon dioxide (CO 2 ; refs. 1, 6, 20-24). Both neutrophils and macrophages have been shown to elicit nitration reactions through myeloperoxidase (MPO) activity (15-18). Catalysis of nitrite oxidation by heme...

Quenching of superoxide radicals by green fluorescent protein

“…While no changes in fluorescence was observed when the protein was exposed to O 2 •− , other species of ROS such as hydrogen peroxide or peroxynitrite anion are known to decrease GFP fluorescence in the presence of mercaptoethanol, suggesting that the protein itself was irreversibly damaged [34]. Since peroxynitrite is very reactive under physiological conditions and formed by the reaction between nitric oxide (NO•) and O 2 •− , the presence of high concentrations of GFP in corals would not only be useful in preventing the formation of hydroxyl radicals but also in preventing the formation of peroxynitrite since NO• is known to occur in symbiotic cnidarians and nitric oxide synthase activity increases in corals experiencing thermal stress [35,36].…”

Transmembrane Nitration of Hydrophobic Tyrosyl Peptides

“…The relevant rate constants of peroxynitrite and ⅐ NO 2 with ⅐ NO, tyrosine, CO 2 , and thiols are taken from Ref. 46. peroxidatic oxidation of nitrite anion diffuses into cells and nitrates a tyrosyl residue present within a green fluorescent protein (52). Nitration of fluorophore quenches the intrinsic green fluorescent protein fluorescence because of an electronwithdrawing effect of the nitro group.…”

Reactions of PTIO and Carboxy-PTIO with ·NO, ·NO2, andO2.¯

“…In addition, Akaike et al (12) demonstrated that excess of ⅐ NO 2 had no effect on the EPR spectrum of PTIOs and, subsequently, Reaction 1 has been adopted to generate ⅐ NO 2 (19,20). Likewise, direct scavenging of ⅐ NO by PTIOs has been shown to inhibit endothelium-derived relaxing factor (EDRF) in bioassay in vitro (12), to cause cell cycle alteration, to result in oxidative stress and apoptosis in pulmonary cells (21), and to exhibit a potent therapeutic effect in cases of endotoxin shock (22).…”