Peroxynitrite-mediated inactivation of heme oxygenases

BMC Pharmacology

Table 2 Time dependent reversibility of the effect of ONOO^- and DEA-NONOate on the catalytic activity of HO-1 (spleen microsomes) and HO-2 (brain microsomes)

		Heme oxygenase activity (pmoles CO/min/mg protein) at different pre-incubation times
Experimental conditions		20 min	60 min	120 min
Spleen microsomes (HO-1)	ONOO^-
	Control	10.4 ± 1.0	10.8 ± 0.8	8.7 ± 0.9
	0.2 mM	3.6 ± 1.4*	10.5 ± 0.8	8.3 ± 1.1
	DEA-NONOate
	Control	10.6 ± 2.0	11.5 ± 1.3	9.4 ± 0.2
	0.2 mM	6.6 ± 0.7*	11.1 ± 1.4	8.9 ± 0.3
Brain microsomes (HO-2)	ONOO^-
	Control	3.6 ± 0.5	4.2 ± 0.4	4.4 ± 0.3
	2 mM	1.9 ± 0.1*	4.4 ± 0.3	3.8 ± 0.3
	DEA-NONOate
	Control	3.6 ± 0.3	4.1 ± 0.3	4.0 ± 0.3
	2 mM	3.0 ± 0.3	3.9 ± 0.1	3.6 ± 0.3

Microsomal protein (50–100 μg) was incubated with 0.2 mM or 2 mM (final concentration) of ONOO^- and DEA-NONOate in 100 mM potassium phosphate buffer, pH 7.4, at 37°C for 20, 60 and 120 minutes. The reaction was stopped by dilution of the reaction mixture to a protein concentration of (0.5 mg/mL) in 100 mM phosphate buffer containing 1 mM NADPH and 50 μM methemalbumin. Incubations of the pretreated microsomal fractions were done as described in materials and methods. Data are presented as the mean ± SD of triplicate experiments. Asterisks denote significant inhibition of the respective HO activity using a one-way ANOVA, P ≤ 0.05.