TABLE 2 Formation of metHb from oxyHb by GTN-derived NO
WT or mutant ALDH2 (4.5 μM) was incubated for 20 min in 0.25 ml of potassium phosphate (50 mM, pH 7.4) containing 1000 U/ml SOD, 10 U/ml catalase, 0.1 mM DTPA, 1 mM NAD+, 3 mM MgCl2, and 10 μM oxyHb in the absence or presence of 2 mM DTT, followed by addition of 0.1 mM GTN at 37°C. Blank values were determined in the absence of protein under otherwise identical conditions and subtracted. Curves were fitted as explained under Materials and Methods to estimate fast and slow contributions to NO formation. Please note that in many instances, especially in the presence of DTT, NO was still being formed at the end of the incubation, so that [NO]slow and the NO/ALDH stoichiometry merely represent the values observed after 20 min. Results are mean values ± S.E. determined in three to five independent experiments.