The specificity of nitroxyl chemistry is unique among nitrogen oxides in biological systems

Abstract

The importance of nitric oxide in mammalian physiology has been known for nearly 30 years. Similar attention for other nitrogen oxides such as nitroxyl (HNO) has been more recent. While there has been speculation as to the biosynthesis of HNO, its pharmacological benefits have been demonstrated in several pathophysiological settings such as cardiovascular disorders, cancer, and alcoholism. The chemical biology of HNO has been identified as related to, but unique from, that of its redox congener nitric oxide. A summary of these findings as well as a discussion of possible endogenous sources of HNO is presented in this review.

FIG. 1.

Indirect high-performance liquid chromatography detection of HNO formation by sulfinamide production. The reaction was performed in phosphate-buffered saline (pH 7.4) with 50 μM of the metal chelator diethylene triamine pentaacetic acid (DTPA) for 10 min at 37°C. All reagents were present at 100 μM. Samples were filtered prepared and analyzed using the methodology described in (34). Briefly, samples (250 μL) were separated on a Kromasil C-18 column (250×4.6 mm, 5 μm particle size, equipped with a guard column; Phenomenex, St. Torrance, CA) by a step gradient from buffer A (pairing reagent 10 mM tetrabutylammonium hydroxide, 10 mM KH₂PO₄, and 0.25% methanol, pH 7.00) to buffer B (2.8 mM tetrabutylammonium hydroxide, 100 mM KH₂PO₄, and 30% methanol, pH 5.50) as follows: 10 min of 100% buffer A, 3 min at up to 80% buffer A, 10 min at up to 70% buffer A, and 12 min at up to 55% buffer A. A flow rate of 1.2 ml/min, column temperature of 18°C, and sample temperature of 4°C were employed. The wavelength of detection was 208 nm except for GSNO, which was 334 nm. Ultrapure standards of GSH, GSSG, nitrite, and nitrate were freshly prepared and used to identify eluted peaks by comparison of retention times and absorption spectra. All samples were filtered through a Centricon MW 3000 cutoff filter before high-performance liquid chromatography analysis. (A) Angeli's salt and GSH, (B) Angeli's salt and oxidized glutathione (GSSG), (C) Angeli's salt and a mixture of GSH/GSSG, (D) DEA/NO and GSH, (E) DEA/NO and GSSG, and (F) DEA/NO and a mixture of GSH/GSSG. Sulfinamide formation was observed only in samples containing Angeli's salt and GSH (A, C). DEA/NO reacted with GSH to produce GSNO (D, F). HNO, nitroxyl; GSH, glutathione; GSNO, S-nitrosogluathione; GSSG, glutathione disulfide; DEA/NO, diethylamine NONOate. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 2.

Indirect electrochemical detection of HNO formation by hydroxylamine production. Real-time detection of NH₂OH was performed using an H₂O₂ electrode from WPI on an Apollo 4000 series instrument. We found that this electrode is far more sensitive to NH₂OH than to H₂O₂ (data not shown). NH₂OH was detected in the presence of 100 μM catalase to eliminate interference from H₂O₂. The reaction conditions were as described in Figure 1 except that 1 mM GSH was exposed to varied concentrations of Angeli's salt (0–100 μM). NH₂OH, hydroxylamine; H₂O₂, hydrogen peroxide.

FIG. 3.

Reactivity of HNO with Cu,Zn SOD and MnSOD. Real-time measurement of NO produced from Angeli's salt (5 μM) and upon the addition of Cu,Zn SOD or MnSOD (20 μM) in the presence of GSH (100 μM), as in Figure 1 using an NO-specific electrode (ISO-NOP, WPI Apollo 4000 series instrument). In the presence of Cu,Zn SOD, there was rapid conversion of HNO to NO. In contrast, the HNO-MnSOD complex slowly degraded. SOD, superoxide dismutase. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 4.

Cyclic Voltammetry measurements of suberoylanilidehydroxamic acid. The voltammogram was obtained with an EG Potensiostat/Galvanostat Model 273A from AMETEK Princeton Applied Research (Oak Ridge, TN). Measurements were performed on 2 mM suberoylanilidehydroxamic acid under aerobic conditions at room temperature in acetonitrile (2 mM) using the platinum auxiliary electrode and Ag/AgCl (saturated KCl) reference electrode.

FIG. 5.

Peroxidation of NH₂OH by heme proteins [adapted from (33)]. , nitrate, GSNHOH, glutathione N-hydroxysulfenamide; GS(O)NH₂, sulfinamide. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 6.

Reactivity of HNO with HRP and catalase. Detection of NO produced from the aerobic reaction of NH₂OH (500 μM) and H₂O₂ (50 μM, added last to initiate the reaction) in the presence of HRP (100 μM) or catalase (100 μM) as in Figure 1 using an NO-specific electrode (ISO-NOP, WPI Apollo 4000 series instrument). The two profiles show the higher production of NO from HRP (500 nM at 20 min) compared with catalase (250 nM at 20 min). HRP, horseradish peroxidase. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).