Peroxynitrite-mediated inactivation of heme oxygenases

BMC Pharmacology

Table 1 Effect of NO donors and sulfhydryl modifying reagents on the catalytic activity of HO-1 (spleen microsomes) and HO-2 (brain microsomes)

	Heme oxygenase activity (pmoles CO/min/mg protein)
Experimental conditions	HO-1 (Spleen microsomes)	HO-2 (Brain microsomes)
Control	11.9 ± 0.6	3.1 ± 0.5
ONOO^-
0.2 mM	5.4 ± 0.9*	2.7 ± 0.6
2 mM	3.8 ± 0.9*	1.4 ± 0.3*
GSNO
0.2 mM	6.5 ± 0.3*	2.9 ± 0.5
2 mM	4.2 ± 0.2*	1.4 ± 0.1*
DEA-NONOate
0.2 mM	4.7 ± 0.4*	2.9 ± 0.7
2 mM	3.6 ± 0.4*	2.4 ± 0.5
NEM
0.2 mM	8.7 ± 0.8	2.9 ± 0.3
2 mM	3.4 ± 0.5*	2.6 ± 0.4
H₂O₂
0.2 mM	11.3 ± 0.2	3.0 ± 0.4
2 mM	11.8 ± 1.5	3.2 ± 0.6
ONOO^- and GSNO
0.2 mM	4.3 ± 0.6*	2.8 ± 0.4
2 mM	4.0 ± 0.2*	1.3 ± 0.2*
(ONOO^- and DEA-NONOate)
0.2 mM	4.7 ± 0.1*	2.6 ± 0.2
2 mM	3.9 ± 0.6*	1.4 ± 0.3*

Microsomal protein (50–100 μg) was incubated with 0.2 mM or 2 mM (final concentration) of ONOO^-, GSNO, DEA-NONOate, NEM, H₂O₂, equimolar concentrations of ONOO^- and GSNO or ONOO^- and DEA-NONOate in 100 mM potassium phosphate buffer, pH 7.4, at 37°C for 20 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 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. Asterisks denote significant inhibition of the respective HO activity using a one-way ANOVA, P ≤ 0.05.