Optimization of purified cDNA-expressed P450 2A6 coumarin 7-hydroxylase activity
| Reconstitution Conditions | Relative Percent Activity2-a |
|---|---|
| Complete reaction mixture2-b | 100 |
| (−) NADPH-GS | 1 |
| (−) 12.5 μg DLPC (12.5 μg total) | 100 |
| (+) 25 μg DLPC (50 μg total) | 84 |
| (+) 50 mM potassium phosphate (pH 7.4) (75 mM total) | 66 |
| 1:0:12-c | 3 |
| 1:1:1 | 62 |
| 1:3:1 | 89 |
| 1:4:1 | 89 |
| 1:5:1 | 95 |
| 1:2:0 | 46 |
| 1:2:2 | 100 |
| 1:2:3 | 101 |
↵2-a The substrate used to optimize P450 2A6 activity was coumarin (25 μM), and the rate of 7-hydroxycoumarin formation obtained with the complete reaction mixture (19.4 nmol/nmol P450/min) was defined as 100%.
↵2-b Complete reaction mixture was composed of a P450 2A6:rat P450 reductase:human cytochrome b5molar ratio of 1:2:1, 25 mM potassium phosphate buffer (pH 7.4), 25 μg of DLPC, and the NADPH-GS.
↵2-c Ratios represent molar ratios of P450 2A6:rat P450 reductase:human cytochrome b5 in 25 mM potassium phosphate buffer (pH 7.4), 25 μg of DLPC, and the NADPH-GS.