CHED 916 |
| The routinely used approach to measure the activity of cytochrome P450 3A4 (CYP3A4) is based on a micellar reconstituted system with phospholipids and cytochrome P450 reductase (CPR). In this study we sought to develop and optimize conditions for reconstitution of a soluble lipid free reconstituted system with a nonionic detergent in order to study protein-protein interactions of CYP3A4. Optimization for the lipid free system included varying the protein concentration, the detergent concentration and the temperature. Based on the results, the ideal conditions were chosen to be 0.05% Triton N-101 reduced, 0.5 µM enzyme and a temperature of 37ºC. Using this new reconstituted system the dissociation constant for the interaction of CYP3A4 with CPR was determined to be 0.43 ?M with a maximal turnover number for 7-BFC debenzylation of 0.5 nmol/min/nmol. Subsequently we found that the addition of cytochrome b5 doubled the activity of 3A4, and a decrease in ionic strength results in higher activity. Our results show this system to be useful in the studies of protein-protein interactions. [Supported by NIH GM54995]. |
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Undergraduate Research Poster Session: Biochemistry
2:00 PM-4:00 PM, Monday, March 26, 2007 Hyatt Regency Chicago -- Riverside Center, Poster
Division of Chemical Education |