THE NITRIC-OXIDE SUPEROXIDE ASSAY - INSIGHTS INTO THE BIOLOGICAL CHEMISTRY OF THE NO O-2(-CENTER-DOT) INTERACTION/

Nitric oxide (NO) is a widespread signaling molecule involved in the r egulation of an impressive spectrum of diverse cellular functions. Sup eroxide anions (O-2(radical anion)) not only contribute to the localiz ation of NO action by rapid inactivation, but also give rise to the fo rmation of the potentially toxic species peroxynitrite (ONOO-) and oth er reactive nitrogen oxide species. The chemistry and biological effec t of ONOO- depend on the relative rates of formation of NO and O-2(rad ical anion). However, the simultaneous quantification of NO and O-2(ra dical anion) has not been achieved yet due to their high rate of inter action, which is almost diffusion-controlled. A sensitive spectrophoto metric assay was developed for the simultaneous quantification of NO and O-2(radical anion) in aqueous solution that is based on the NO-ind uced oxidation of oxyhemoglobin (oxyHb) to methemoglobin and the O-2(r adical anion)-mediated reduction of ferricytochrome c. Using a photodi ode array photometer, spectral changes of either reaction were analyze d, and appropriate wavelengths were identified for the simultaneous mo nitoring of absorbance changes of the individual reactions, oxyHb oxid ation was followed at 541.2 nm (isosbestic wavelength for the conversi on of ferri- to ferrocytochrome c), and ferricytochrome c reduction wa s followed at 465 nm (wavelength at which absorbance changes during ox yHb to methemoglobin conversion were negligible), using 525 nm as the isosbestic point for both reactions, At final concentrations of 20 mu M ferricytochrome c and 5 mu M oxyHb, the molar extinction coefficient s were determined to be epsilon(465-525) = 7.3 mM(-1) cm(-1) and epsil on(541.2-525) = 6.6 mM(-1) cm(-1), respectively. The rates of formatio n of either NO or O-2(radical anion) determined with the combined assa y were virtually identical to those measured with the classical oxyhem oglobin and cytochrome c assays, respectively. The assay was successfu lly adapted to either kinetic or end point determination in a cuvette or continuous on-line measurement of both radicals in a flow-through s ystem. Maximal assay sensitivity was -25 nM for NO and O-2(radical ani on). Cross-reactivity with ONOO- was controlled for by the presence (i f L-methionine. Generation of NO from the NO donor spermine diazeniumd iolate could be reliably quantified in the presence and absence of low , equimolar, and high flux rates of O-2(radical anion). Likewise, O-2( radical anion) enzymatically generated from hypoxanthine/xanthine oxid ase could be specifically quantified with no difference in absolute ra tes in the presence or absence of concomitant NO generation at differe nt flux rates. Nonenzymatic decomposition of 3-morpholinosydnonimine h ydrochloride (100 mu M) in phosphate buffer, pH 7.4 (37 degrees C), wa s found to be associated with almost stoichiometric production of NO a nd O-2(radical anion) (1.24 mu M NO/min and 1.12 mu M O-2(radical anio n)/min). Assay selectivity and applicability to biological systems wer e demonstrated in cultured endothelial cells and isolated aortic tissu e using calcium ionophore and NADH for stimulation of NO and O-2(radic al anion) formation, respectively. Based on these data, a computer mod el was elaborated that successfully predicts the reaction of NO and O- 2(radical anion) with hemoprotein and may thus help to further elucida te these reactions. In conclusion, the nitric oxide/superoxide assay a llows the specific, sensitive, and simultaneous detection of NO and O- 2(radical anion). The simulation model developed also allows the relia ble prediction of the reaction between NO and O-2(radical anion) as we ll as their kinetic interaction with other biomolecules. These new ana lytical tools will help to gain further insight into the physiological and pathophysiological significance of the formation of these radical s in cell homeostasis.