Kinetic Analysis of Intracellular Concentrations of Reactive Nitrogen Species

Abstract

Reactive nitrogen species derived from NO have been implicated in cancer and other diseases, but their intracellular concentrations are largely unknown. To estimate them under steady-state conditions representative of inflamed tissues, a kinetic model was developed that included the effects of cellular antioxidants, amino acids, proteins, and lipids. For an NO concentration of 1 µM, total peroxynitrite (Per, the sum of ONOO− and ONOOH), NO2·, and N2O3 were calculated to have concentrations in the nM, pM, and fM ranges, respectively. The concentrations of NO2· and N2O3 were predicted to decrease markedly with increases in glutathione (GSH) levels, due to the scavenging of each by GSH. Although lipids accelerate the oxidation of NO by O2 (because of the high solubility of each in hydrophobic media), lipid-phase reactions were calculated to have little effect on NO2· or N2O3 concentrations. The major sources of intracellular NO2· were found to be the reaction of Per with metals and with CO2, whereas the major sinks were its reactions with GSH and ascorbate (AH−). The radical-scavenging ability of GSH and AH− caused 3-nitrotyrosine to be the only tyrosine derivative predicted to be formed at a significant rate. The major GSH reaction product was S-nitrosoglutathione. Analytical (algebraic) expressions are provided for the concentrations of the key reactive intermediates, allowing the calculations to be extended readily.

Comparative and integrative metabolomics reveal that S-nitrosation inhibits physiologically relevant metabolic enzymes

“…COMT assay: The activity assay buffer was 100 mM sodium phosphate at pH 7.6 and 1.2 mM MgCl 2 . The assay mixture contained 1 mM SAM with varying concentrations 3,4-dihydroxybenzoic acid (DHBA) (5,10,15,20,40,60 or 100 µM) and was pre-incubated for 2-3 min at 37 °C before enzyme addition. The assay was initiated by the addition of 200 nM COMT in a 450 µL Eppendorf tube and incubated at 37 °C.…”

“…GSNO stock concentrations were measured before use (ε 335 nm = 0.992 mM -1 cm -1 ). All proteins, except PHGDH, were incubated separately at 20 µM with varying GSNO or GSH concentrations (20,50, 100, 150, 200, 500, 1000 or 2000 µM) at 37 °C for 1 h. PHGDH was incubated at 150 µM with GSNO or GSH concentrations of 150, 500, 1000 or 2000 µM for 1 h. For kinetics experiments, 6PGD and PHGDH were incubated with 2 mM GSH or GSNO for 1 h at 37 ˚C, ALDH4A1, ALDOA, and TPI1 were incubated with 1 mM GSH or GSNO for 1 h at 37˚C and COMT was incubated for 150 µM GSH or GSNO for 1 h at 37 ˚C. Proteins were spun at ~21,000 rcf for 1 min to remove precipitation and the supernatant was buffer exchanged into the appropriate activity buffer using Bio-Spin6 columns.…”

Specificity in S-Nitrosylation: A Short-Range Mechanism for NO Signaling?

“…Kinetic models integrating these reactions with the formation of other RNS have shown that N 2 O 3 formation would not be very important at the NO concentrations estimated for biological systems (82,87).…”

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease

“…NO reacts with superoxide anion (O 2 −• ) at diffusion-controlled rates to yield highly reactive peroxynitrite (ONOO − ) (26,27). MPO also reacts H 2 O 2 with nitrite (NO 2 − , the endpoint of cellular NO oxidation) to produce the strong nitrating agent, nitrogen dioxide radical (NO 2 • ) (28).…”