Table 2.

Remaining catalytic acitivity and heme content of cytochrome P450 2E1 and 2E1 T303A after incubation with reductase.

Wild-type P450 2E1 and T303A mutant were incubated for 10 min with reductase in the absence and presence of 1mM NADPH and in the presence of NADPH and 2000 units/ml catalase or 200 mM 4MP as described under Experimental Procedures. After incubation for 10 min, the 7-EFC O-deethylation activity was measured. Reduced CO-spectra were measured to quantify the P450 content. Heme content and P450 protein recovered were measured by HPLC monitored at 398 nm and 220 nm, respectively. The data represent the mean and standard deviations from three separate incubations.

Primary reaction mixture	% remaining
	Activity		Reduced CO		Heme		P450 Protein recovery
	2E1	T303A	2E1	T303A	2E1	T303A	2E1	T303A
−NADPH without incubation	100	100	100	100	100	100	100	100
−NADPH	91 ± 3	88 ± 6	92 ± 1	93 ± 2	99 ± 1	100 ± 1	100 ± 3	101 ± 1
+NADPH	87 ± 5	53 ± 8	82 ± 5	36 ± 12	80 ± 1	47 ± 6	94 ± 5	67 ± 6
+NADPH, +Catalase	93 ± 2	87 ± 3	88 ± 1	77 ± 2	85 ± 6	93 ± 2	96 ± 4	98 ± 5
+NADPH, +4MP	NA	NA	101 ± 2	93 ± 5	88 ± 3	93 ± 3	96 ± 5	102 ± 5

NA: not able to measure catalytic activities due to the strong competitive inhibiton by 4MP.