Fig. 5.

GSH does not react directly with the NONOates. (A) Effect of oxy-Hb on the GSNO yield from 1 µM DEA/NO and 1 mM GSH. In the presence of oxy-Hb (1 µM; 10 s) the NO peak observed after addition of DEA/NO (1 µM; 90 s) was very small, and no GSNO formation was evident after CuSO₄ addition (4 mM; 506 s), whereas subsequent GSNO administration (2 µM; 570 s) yielded a pronounced peak. Experimental conditions: 1 mM GSH, 1000 U/ml SOD, 0.1 mM DTPA, 5 mM MgCl₂, and 50 mM TEA (pH 7.4) in 0.5 ml at 37 °C; at the indicated times 1 µM oxy-Hb, 1 µM DEA/NO, 4 mM CuSO₄, and 2 µM GSNO were added. (B) Effect of the time of GSH administration on the yield of GSNO from PROLI/NO and GSH. In the presence of 1 mM GSH the NO peak derived from 1 µM PROLI/NO was considerably smaller than in the absence of GSH, and a strong NO signal, originating from GSNO, was observed after CuSO₄ addition (compare the dotted and dashed traces in the absence and presence of GSH, respectively). If GSH was added 50 s after PROLI/NO, at a time when all PROLI/NO should be decomposed, CuSO₄ addition still caused a sizeable NO signal (continuous trace). Experimental conditions: 1 µM PROLI/NO, 1000 U/ml SOD, 0.1 mM DTPA, 4 mM CuSO₄, 5 mM MgCl₂, and 50 mM TEA (pH 7.4) in 0.5 ml at 37 °C; GSH (1 mM) was absent (dotted trace), present (dashed trace), or added at the indicated time (continuous trace).