Abstract
Nitric oxide (NO) is a widespread signaling molecule involved in the regulation of an impressive spectrum of diverse cellular functions. Superoxide anions (O-2) not only contribute to the localization of NO action by rapid inactivation, but also give rise to the formation of the potentially toxic species peroxynitrite (ONOO-) and other reactive nitrogen oxide species. The chemistry and biological effect of ONOO- depend on the relative rates of formation of NO and O-2. However, the simultaneous quantification of NO and O-2 has not been achieved yet due to their high rate of interaction, which is almost diffusion-controlled. A sensitive spectrophotometric assay was developed for the simultaneous quantification of NO and O-2 in aqueous solution that is based on the NO-induced oxidation of oxyhemoglobin (oxyHb) to methemoglobin and the O-2-mediated reduction of ferricytochrome c. Using a photodiode array photometer, spectral changes of either reaction were analyzed, and appropriate wavelengths were identified for the simultaneous monitoring of absorbance changes of the individual reactions. oxyHb oxidation was followed at 541.2 nm (isosbestic wavelength for the conversion of ferri- to ferrocytochrome c), and ferricytochrome c reduction was followed at 465 nm (wavelength at which absorbance changes during oxyHb to methemoglobin conversion were negligible), using 525 nm as the isosbestic point for both reactions. At final concentrations of 20 microM ferricytochrome c and 5 microM oxyHb, the molar extinction coefficients were determined to be epsilon465-525 = 7.3 mM-1 cm-1 and epsilon541.2-525 = 6.6 mM-1 cm-1, respectively. The rates of formation of either NO or O-2 determined with the combined assay were virtually identical to those measured with the classical oxyhemoglobin and cytochrome c assays, respectively. The assay was successfully adapted to either kinetic or end point determination in a cuvette or continuous on-line measurement of both radicals in a flow-through system. Maximal assay sensitivity was approximately 25 nM for NO and O-2. Cross-reactivity with ONOO- was controlled for by the presence of L-methionine. Generation of NO from the NO donor spermine diazeniumdiolate could be reliably quantified in the presence and absence of low, equimolar, and high flux rates of O-2. Likewise, O-2 enzymatically generated from hypoxanthine/xanthine oxidase could be specifically quantified with no difference in absolute rates in the presence or absence of concomitant NO generation at different flux rates. Nonenzymatic decomposition of 3-morpholinosydnonimine hydrochloride (100 microM) in phosphate buffer, pH 7.4 (37 degrees C), was found to be associated with almost stoichiometric production of NO and O-2 (1.24 microM NO/min and 1.12 microM O-2/min). Assay selectivity and applicability to biological systems were demonstrated in cultured endothelial cells and isolated aortic tissue using calcium ionophore and NADH for stimulation of NO and O-2 formation, respectively. Based on these data, a computer model was elaborated that successfully predicts the reaction of NO and O-2 with hemoprotein and may thus help to further elucidate these reactions. In conclusion, the nitric oxide/superoxide assay allows the specific, sensitive, and simultaneous detection of NO and O-2. The simulation model developed also allows the reliable prediction of the reaction between NO and O-2 as well as their kinetic interaction with other biomolecules. These new analytical tools will help to gain further insight into the physiological and pathophysiological significance of the formation of these radicals in cell homeostasis.
Highlights
From the ‡Department of Medicine, Division of Cardiology, Pulmonary Diseases, and Angiology, Heinrich Heine University, Moorenstrasse 5, D-40225 Dusseldorf, Federal Republic of Germany, and the §Tumor Biology Section, Radiation Biology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892-1002
As pilot tests revealed that the presence of oxyHb causes slow, superoxide dismutase-independent reduction of ferricytochrome c over time, all measurements for direct comparison of the “classical” cytochrome c assay with the combined Nitric oxide (NO)/O2. assay were performed in the presence of 5 M oxyHb
We have shown that the reoxidation of ferrocytochrome c in the presence of fluxes of NO/O2. is abolished by methionine, indicating that ONOOϪ is the oxidant responsible for this reaction
Summary
Vol 272, No 15, Issue of April 11, pp. 9922–9932, 1997 Printed in U.S.A. (Received for publication, October 16, 1996, and in revised form, February 3, 1997). In contrast to the current view that NO is extremely unstable in vivo, but in agreement with its functioning as a paracrine mediator, NO can travel significant distances to reach target cells neighboring the NO-generating cell [4, 5] Along this migration, in particular at higher concentration, NO can interact with molecular oxygen to form higher nitrogen oxides (e.g. NO2 and N2O3), which can either react with other biomolecules such as thiols and amines or hydrolyze to form nitrite (NO2Ϫ) and nitrate (NO3Ϫ). Are formed, almost all methods for measurement of either molecule can detect only that amount of radical that escaped from chemical interaction with the respective other reaction partner (and, other biomolecules), leading to significant underestimation of their true rates of formation. The results obtained with this new assay could be successfully simulated by a computer model that predicts the reaction of both radicals with hemoproteins and may give valuable new insights into the reaction between NO and O2. in biological systems
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