Fig. 3.
Effects of GSH concentration on the NO decay rate and the GSNO yield in the absence and presence of 5 mM MgCl2. (A) Apparent pseudo-first-order rate constants (observed at 0.75 µM NO) as a function of the GSH concentration. The lines are best linear fits (y=ax), with fitting parameters a, representing the apparent second-order rate constants for the reaction between NO and GSH, of 8.8±0.4 M−1 s−1 (R=0.989) and 0.52±0.13 M−1 s−1 (R=0.82) in the presence and absence of Mg2+, respectively. (B) GSNO yields measured as NO released after injection of CuSO4. The lines through the data points are best fits to the hyperbola y=bx/(a+x), with a and b representing the EC50 for GSH and the maximal yield of GSNO, respectively. Fitting parameters: in the presence of Mg2+, EC50 0.72±0.06 mM, [GSNO]max 0.91±0.03 µM (R=0.992); in the absence of Mg2+, EC50 0.34±0.06 mM, [GSNO]max 0.40±0.03 µM (R=0.991). Experimental conditions: 1 µM PROLI/NO, varying concentrations of GSH (0.1–5 mM), 4 mM CuSO4, 1000 U/ml SOD, 0.1 mM DTPA, 0 (white circles, dotted line) or 5 (black circles, continuous line) mM MgCl2, and 50 mM TEA (pH 7.4) at 37 °C. Also included are the results obtained for 2 mM GSH with authentic NO (0.75 mM) in the absence (white squares) and presence (black squares) of 5 mM MgCl2. Data are shown ±SEM (n=5).