FIGURE 4.

Superoxide and peroxynitrite formation from increasing concentrations of purified ALDH-2 in the presence and absence of GTN. A, ALDH-2 (6.9 μm stock based on the monomers (= 386 μg/ml) diluted 1:100–1:3000) was incubated for 5 min with NAD⁺ (100 μm) and either lucigenin (Luci, 100 μm) or L-012 (100 μm) in PBS (Dulbecco's phosphate-buffered saline, without DTT, Ca²⁺/Mg²⁺) at room temperature. Where indicated, 1 μm GTN was added. RONS formation was detected over 5 min and expressed as counts/min (relative light units (RLU)) at 5 min. B, conditions as described for panel A, but only the L-012 data are shown in the presence or absence of PEG-SOD (1000 units/ml). In some experiments, BSA (0–69 nm) was used instead of ALDH-2. Data are mean ± S.E. of three (L-012) or five (lucigenin) independent experiments. ROS, reactive oxygen species.