Fig. 13. Effect of desferal and cysteine on the decomposition of Cys-NO as monitored by the optical absorption at 340 nm. The kinetics was studied at room temperature in HEPES open to ambient air (15 mM, pH 7.4). Panel A: (1) decomposition of 2 mM Cys-NO without additives; (2) decomposition of 1.5 mM Cys-NO in presence of 0.5 mM cysteine; (3) 2 mM Cys-NO in presence of 1 mM desferal; (4) 2 mM Cys-NO in presence of 1 mM cysteine. Panel B: (1) 2 mM Cys-NO in presence of 0.1 mM cysteine; (2) 2 mM Cys-NO in presence of 0.25 mM cysteine. (From Ref. .)
of 2 mM Cys-NO in HEPES buffer (15 mM, pH 7.4) open to ambient air and at room temperature.
At these conditions, the oxygen concentration is ca 0.25 mM, and the decomposition of Cys-NO was completed within several minutes (Fig. 13, Panel A curves 1,2). The decomposition was inhibited by 1 mM iron chelator desferal or by 2 mM cysteine (Fig. 13, Panel A
curves 3,4). Cysteine was found to inhibit only at concentrations significantly higher than the oxygen concentration of 0.25 mM. At lower doses, Cys-NO was stabilized only for a short period of time after which rapid decomposition took place (Fig. 13, Panel B curves 1,2).
In the absence of excess free cysteine, the decomposition of 2 mM Cys-NO caused the loss of oxygen from the solution (Fig. 14, curve 1). This consumption of oxygen was halted if the decomposition of Cys-NO was inhibited by either desferal or excess cysteine (Fig. 14, curve 2). Cysteine by itself did not detectably affect the oxygen concentration in HEPES buffer. In the presence of Cys-NO and excess cysteine ligand, a small quantity of ca 1 ^M Cys-DNIC was detected by EPR on frozen aliquots of the buffer solution. Since no iron had been added, the DNIC was attributed to the pool of spurious iron in the solutions and reaction vessels. The chelation experiment with desferal shows that this pool of spurious iron was responsible for the observed decomposition of Cys-NO.
The observations are interpreted by the following hypothesis : In oxygenated solutions, the spurious iron is largely ferric. In the absence of free cysteine, the decomposition of Cys-NO is initiated by oxidation by ferric iron as shown in Scheme 10. This scheme is similar to Scheme 2 for the oxidative mechanism of RS-NO decomposition catalyzed by copper. The oxygen rapidly restores the iron to ferric state by oxidation
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