Table 2. Spectrophotometric analysis of O₂^•−-scavenging activity in intact mitochondria.

O₂^•−-scavenging activity of mitochondria was assessed by following the reduction of either XTT (470 nm) or acetylated cytochrome c (550 nm). The reaction mixtures consisted of 20 m-units/ml xanthine oxidase, 50 μM xanthine, and 750 μM XTT or 10 μM acetylated cytochrome c in either sucrose/Tris buffer (in the absence of KCN) or phosphate buffer (50 mM potassium phosphate and 0.1 mM EDTA, pH 7.8) (in the presence of KCN). Where indicated, the following were added to the reaction mixture: bovine Cu,Zn-superoxide dismutase (0.4 μM); intact mitochondria (100 μg of protein/ml); intermembrane space preparation (25 μg of protein/ml); KCN (5 mM). KCN at a concentration of 20 μM was added to the reaction mixture described in the third column in order to inhibit cytochrome c oxidase activity. n.d., not determined.

	XTT reduction (μM/min)		Acetylated cytochrome c reduction (μM/min)
Assay conditions	−KCN	+KCN	−KCN	+KCN
No additions	0.57±0.04	0.60±0.05	1.26±0.10	1.30±0.12
Mitochondria	0.55±0.04	n.d.	1.20±0.10	n.d.
Intermembrane space	0.33±0.03	0.56±0.05	0.65±0.06	1.21±0.10
Bovine Cu,Zn-SOD	0.005±0.0001	0.48±0.04	0.01±0.0002	1.10±0.10