FIGURE 4.

Effect of sodium dithionite on the formation of CO-reduced Hmp upon addition of CORM-3 in anaerobic conditions. Hmp (5 μm) was suspended in 50 mm Tris-HCl, 50 mm NaCl buffer (pH 8.0) containing NADH (500 μm) anaerobically. Arrow (A to B) indicates subsequent steps performed with the same sample. Reduction of Hmp was promoted by 15 min incubation in NADH-containing buffer (solid line in A) followed by the addition of 10 μm CORM-3. Changes were recorded after 30 min incubation (dashed line in A and B). An excess of sodium dithionite was added, and changes in the spectra were recorded every 0.5 min for 12 min (gray lines in B). C, difference spectra after addition of dithionite. D, CO release time course from CORM-3 before and after addition of dithionite. Dashed arrows indicate the direction of the changes. For the absolute spectrum of reduced Hmp (A), NADH-containing buffer was used as baseline. The experiment was repeated three times with similar results.