Figure 1

ONOO^--mediated inactivation of HO-1 and HO-2. Rat spleen (Figure 1A) and brain microsomes (Figure 1B) (50–100 μg protein) were treated with indicated concentrations of ONOO^- or degraded ONOO^- in 100 mM potassium phosphate, pH 7.4 at room temperature for 10 seconds. The reaction was stopped by dilution of the reaction mixture to a protein concentration of (0.5 mg/mL) spleen microsomes and (1 mg/mL) brain microsomes in 100 mM phosphate buffer containing 1 mM NADPH and 50 μM methemalbumin. Incubations of the pretreated microsomal fractions were performed for 15 min and enzyme activity was determined by the quantitation of CO formed in the reaction mixture. Data are presented as the mean ± SD of triplicate experiments. The rates of CO formation in the control reactions were 12 ± 1 and 5 ± 1 pmoles CO/min/mg protein for spleen and brain microsomes respectively. IC₅₀ values for HO-1 and HO-2 were 0.015 ± 0.005 mM and 1.25 ± 0.25 mM respectively.