First P450 2B6 was incubated with Isoimperatorin glutathione

First P450 2B6 was incubated with Isoimperatorin, glutathione and NADPH in potassium phosphate butter (pH 7.4). The total volume was 0.2 mL This primary mixture was preincubated at 30 degree C for 3 min. Some of the primary mixtures were then transferred into the secondary incubation mixtures containing bupropion and NADPH and further incubated at 30 degree C for 30 min, followed by the addition of 0.12 mL ice-cold acetonitrile containing propranolol as internal standard. From the following graph, calculate how effective this inhibitor is by reporting the percent of remaining active enzymes at 9 min from the squarebox plot. Why was the inhibition reaction performed in the present of glutathione (GSH). Why was bupropion added in the second incubation? What the purpose of adding 0.12 mL ice-cold acetonitrile? Why did the author called this type of inhibition of cytochrome P450 2B6 by isoimperatorin a mechanism-based inactivation?

Solution

8. From the graph the plot mentioned in the question has higher inhibition rate in comparison with other inhibitor. The inhibition is 0.4% which is the highest among the inhibitor used.

9. The inhibition is preformed in the presence of GSH because it is reported to protect the p450 enzyme activity

10. The cytochrome p450 activity is measured by the hydroxylation of bupropion, hence it is added in the second inucbation.

11. Ice cold acetonitrile is added to stop the reaction

12. The author called this type of inhibition of cytochrome p450 286 by isoimpertorin a mechanism based inactivation because the inhibitor used is isoimperatorin. and it is evaluated on the overall mechanism of the enzyme.